2024/06/19 更新

写真a

ウエダ タクヤ
上田 卓也
所属
理工学術院 大学院先進理工学研究科
職名
教授(任期付)
学位
農学博士 ( 東京大学 )
農学修士 ( 東京大学 )

経歴

  • 2019年04月
    -
    継続中

    早稲田大学   先進理工学研究科   生命理工学専攻

  • 2019年07月
    -
     

    東京大学   名誉教授

  • 2015年04月
    -
    2019年03月

    東京大学   大学院領域創成科学研究科 メディカル情報生命専攻   教授

  • 2004年04月
    -
    2015年03月

    東京大学   大学院新領域創成科学研究科 メディカルゲノム専攻   教授

  • 2011年04月
    -
    2013年03月

    東京大学   大学院新領域創成科学研究科   研究科長

  • 1999年04月
    -
    2004年03月

    東京大学   大学院新領域創成科学研究科 先端生命科学専攻   教授

  • 1998年04月
    -
    1999年03月

    東京大学   大学院新領域創成科学研究科 先端生命科学専攻   助教授

  • 1996年08月
    -
    1998年03月

    東京大学   大学院工学系研究科 化学・生命工学専攻   助教授

  • 1994年08月
    -
    1996年07月

    東京大学   大学院工学系研究科 化学・生命工学専攻   講師

  • 1992年06月
    -
    1994年07月

    東京大学   工学部 工業化学科   助手

  • 1989年08月
    -
    1992年05月

    東京工業大学   生命理工学部   助手

  • 1988年05月
    -
    1989年07月

    東京工業大学   総合理工学研究科   助手

  • 1986年09月
    -
    1988年04月

    横浜市立大学   木原生物学研究所   助手

  • 1984年05月
    -
    1986年08月

    マックス-プランク実験医学研究所   化学部   博士研究員

▼全件表示

学歴

  • 1981年04月
    -
    1984年03月

    東京大学   大学院農学系研究科   農芸化学専攻博士課程  

  • 1979年04月
    -
    1981年03月

    東京大学   大学院農学系研究科   農芸化学専攻修士課程  

  • 1977年04月
    -
    1979年03月

    東京大学   農学部   農芸化学科  

  • 1974年04月
    -
    1977年03月

    東京大学   理科二類  

所属学協会

  •  
     
     

    日本生物物理学会

  •  
     
     

    日本RNA学会

  •  
     
     

    無細胞生命科学研究会

  •  
     
     

    農芸化学会

  •  
     
     

    生化学会

  •  
     
     

    分子生物学会

▼全件表示

研究分野

  • 構造生物化学 / 機能生物化学 / 進化生物学 / 分子生物学

研究キーワード

  • リボソーム

  • 生体外蛋白質合成系

  • tRNA

  • シャペロン

  • フォールディング

  • 遺伝暗号

  • 抗体

  • コドン

  • アミノアシルtRNA合成酵素

  • rRNA

  • 蛋白質合成系

  • 翻訳

  • 翻訳終結

  • 凝集

  • 変則暗号

  • GroEL

  • 反転膜小胞

  • リボソーム蛋白質

  • 系統樹

  • 進化工学

  • Candida

  • 開始因子

  • 蛋白質相互作用

  • 膜蛋白質

  • カンジダ

  • 分子生物学

  • Molecular Biology

▼全件表示

受賞

  • JB論文賞

    2023年   日本生化学会  

  • JB論文賞

    2012年   日本生化学会  

  • JB論文賞

    1995年   日本生化学会  

 

論文

  • Evaluating the effect of two-dimensional molecular layout on DNA origami-based transporters

    Kodai Fukumoto, Yuya Miyazono, Takuya Ueda, Yoshie Harada, Hisashi Tadakuma

    Nanoscale Advances   5 ( 9 ) 2590 - 2601  2023年

     概要を見る

    Single-molecule fluorescence imaging of DNA origami-based transporters showed shorter run lengths in dence layouts of kinesin motors.

    DOI

    Scopus

    2
    被引用数
    (Scopus)
  • The Escherichia coli DnaK chaperone stimulates the α-complementation of β-galactosidase

    Samuel Berhanu, Takuya Ueda, Jean Hervé Alix

    Journal of Basic Microbiology   62 ( 6 ) 669 - 688  2022年06月

     概要を見る

    pUC18 and pUC19 are well-known high copy-number plasmid vectors routinely used for DNA cloning purposes. We show here that, in Escherichia coli transformed by native pUC18, the α-complementation of β-galactosidase (i.e., mediated by the peptide LacZα18) is intrinsically weak and slow, but is greatly stimulated by the DnaK/DnaJ/GrpE chaperone system. In contrast, the α-complementation mediated by the peptide LacZα19 (in E. coli transformed by the native pUC19) is much more efficient and therefore does not require the assistance of the DnaK chaperone machinery. The marked difference between these two LacZα peptides is reproduced in a cell-free protein expression system coupled with α-complementation. We conclude that: (i) α-complementation of β-galactosidase is DnaK-mediated depending upon the LacZα peptide donor; (ii) DnaK, sensu stricto, is not necessary for α-complementation, but can enhance it to a great extent; (iii) this observation could be used to establish an easy and inexpensive method for screening small molecules libraries in search of DnaK inhibitors and also for deciphering the DnaK-mediated protein quality control mechanism.

    DOI PubMed

    Scopus

  • In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins

    Ryo Aoyama, Keiko Masuda, Masaru Shimojo, Takashi Kanamori, Takuya Ueda, Yoshihiro Shimizu

    Journal of Biochemistry   171 ( 2 ) 227 - 237  2022年02月

     概要を見る

    Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

    DOI PubMed

    Scopus

    8
    被引用数
    (Scopus)
  • Transfer RNA Synthesis and Regulation

    Hiroyuki Hori, Akira Hirata, Takuya Ueda, Kimitsuna Watanabe, Chie Tomikawa, Kozo Tomita

    eLS     1 - 20  2021年11月

    DOI

  • Interleukin-6 sensitizes TNF-α and TRAIL/Apo2L dependent cell death through upregulation of death receptors in human cancer cells

    Emiko Sano, Akira Kazaana, Hisashi Tadakuma, Toshiaki Takei, Sodai Yoshimura, Yuya Hanashima, Yoshinari Ozawa, Atsuo Yoshino, Yutaka Suzuki, Takuya Ueda

    Biochimica et Biophysica Acta - Molecular Cell Research   1868 ( 7 )  2021年06月

     概要を見る

    Interleukin-6 (IL-6) enhanced TNF-α and TRAIL/Apo2L induced cell death in various human cancer cells derived from malignant glioma, melanoma, breast cancer and leukemia, although the effect was not detected with IL-6 alone. The effects of IL-6 using SKBR3 cells were associated with the generation of apoptotic cells as analyzed by fluorescence microscopy and flow cytometry. IL-6 activated p53 and upregulated TRAIL death receptors (DR-4 and DR-5) and stimulated the TNF-α and TRAIL dependent extrinsic apoptotic pathway without activation of the p53 mediated intrinsic apoptotic pathway. TNF-α and TRAIL induced cleavage of caspase-8 and caspase-3 was more enhanced by IL-6, although these caspases were not cleaved by IL-6 alone. The dead cell generation elicited by the combination with IL-6 was blocked by anti-human TRAIL R2/TNFRSF10B Fc chimera antibody which can neutralize the DR-5 mediated death signal. These findings indicate that IL-6 could contribute to the enhancement of TNF-α or TRAIL induced apoptosis through p53 dependent upregulation of DR-4 and DR-5. The data suggest that a favorable therapeutic interaction could occur between TNF-α or TRAIL and IL-6, and provide an experimental basis for rational clinical treatments in various cancers.

    DOI PubMed

    Scopus

    6
    被引用数
    (Scopus)
  • Force measurements show that uL4 and uL24 mechanically stabilize a fragment of 23S rRNA essential for ribosome assembly

    Laurent Geffroy, Thierry Bizebard, Ryo Aoyama, Takuya Ueda, Ulrich Bockelmann

    RNA   25 ( 4 ) 472 - 480  2021年04月  [査読有り]

     概要を見る

    In vitro reconstitution studies have shown that ribosome assembly is highly cooperative and starts with the binding of a few ribosomal (r-) proteins to rRNA. It is unknown how these early binders act. Focusing on the initial stage of the assembly of the large subunit of the Escherichia coli ribosome, we prepared a 79-nucleotide-long region of 23S rRNA encompassing the binding sites of the early binders uL4 and uL24. Force signals were measured in a DNA/RNA dumbbell configuration with a double optical tweezers setup. The rRNA fragment was stretched until unfolded, in the absence or in the presence of the r-proteins (either uL4, uL24, or both). We show that the r-proteins uL4 and uL24 individually stabilize the rRNA fragment, both acting as molecular clamps. Interestingly, this mechanical stabilization is enhanced when both proteins are bound simultaneously. Independently, we observe a cooperative binding of uL4 and uL24 to the rRNA fragment. These two aspects of r-proteins binding both contribute to the efficient stabilization of the 3D structure of the rRNA fragment under investigation. We finally consider implications of our results for large ribosomal subunit assembly.

    DOI PubMed

    Scopus

    3
    被引用数
    (Scopus)
  • Snapshots of native pre-50S ribosomes reveal a biogenesis factor network and evolutionary specialization

    Rainer Nikolay, Tarek Hilal, Sabine Schmidt, Bo Qin, David Schwefel, Carlos H. Vieira-Vieira, Thorsten Mielke, Jörg Bürger, Justus Loerke, Kazuaki Amikura, Timo Flügel, Takuya Ueda, Matthias Selbach, Elke Deuerling, Christian M.T. Spahn

    Molecular Cell   81 ( 6 ) 1200 - 1215.e9  2021年03月

     概要を見る

    Ribosome biogenesis is a fundamental multi-step cellular process that culminates in the formation of ribosomal subunits, whose production and modification are regulated by numerous biogenesis factors. In this study, we analyze physiologic prokaryotic ribosome biogenesis by isolating bona fide pre-50S subunits from an Escherichia coli strain with the biogenesis factor ObgE, affinity tagged at its native gene locus. Our integrative structural approach reveals a network of interacting biogenesis factors consisting of YjgA, RluD, RsfS, and ObgE on the immature pre-50S subunit. In addition, our study provides mechanistic insight into how the GTPase ObgE, in concert with other biogenesis factors, facilitates the maturation of the 50S functional core and reveals both conserved and divergent evolutionary features of ribosome biogenesis between prokaryotes and eukaryotes.

    DOI PubMed

    Scopus

    26
    被引用数
    (Scopus)
  • Reconstitution of mammalian mitochondrial translation system capable of correct initiation and long polypeptide synthesis from leaderless mRNA

    Muhoon Lee, Noriko Matsunaga, Shiori Akabane, Ippei Yasuda, Takuya Ueda, Nono Takeuchi-Tomita

    Nucleic Acids Research   49 ( 1 ) 371 - 382  2021年01月

     概要を見る

    Mammalian mitochondria have their own dedicated protein synthesis system, which produces 13 essential subunits of the oxidative phosphorylation complexes. We have reconstituted an in vitro translation system from mammalian mitochondria, utilizing purified recombinant mitochondrial translation factors, 55S ribosomes from pig liver mitochondria, and a tRNA mixture from either Escherichia coli or yeast. The system is capable of translating leaderless mRNAs encoding model proteins (DHFR and nanoLuciferase) or some mtDNA-encoded proteins. We show that a leaderless mRNA, encoding nanoLuciferase, is faithfully initiated without the need for any auxiliary factors other than IF-2mt and IF-3mt. We found that the ribosome-dependent GTPase activities of both the translocase EF-G1mt and the recycling factor EF-G2mt are insensitive to fusidic acid (FA), the translation inhibitor that targets bacterial EF-G homologs, and consequently the system is resistant to FA. Moreover, we demonstrate that a polyproline sequence in the protein causes 55S mitochondrial ribosome stalling, yielding ribosome nascent chain complexes. Analyses of the effects of the Mg concentration on the polyproline-mediated ribosome stalling suggested the unique regulation of peptide elongation by the mitoribosome. This system will be useful for analyzing the mechanism of translation initiation, and the interactions between the nascent peptide chain and the mitochondrial ribosome.

    DOI PubMed

    Scopus

    13
    被引用数
    (Scopus)
  • Reconstituted cell-free protein synthesis using in vitro transcribed tRNAs

    Keita Hibi, Kazuaki Amikura, Naoki Sugiura, Keiko Masuda, Satoshi Ohno, Takashi Yokogawa, Takuya Ueda, Yoshihiro Shimizu

    Communications Biology   3 ( 1 ) 350 - 350  2020年12月  [査読有り]  [国際誌]

     概要を見る

    Entire reconstitution of tRNAs for active protein production in a cell-free system brings flexibility into the genetic code engineering. It can also contribute to the field of cell-free synthetic biology, which aims to construct self-replicable artificial cells. Herein, we developed a system equipped only with in vitro transcribed tRNA (iVTtRNA) based on a reconstituted cell-free protein synthesis (PURE) system. The developed system, consisting of 21 iVTtRNAs without nucleotide modifications, is able to synthesize active proteins according to the redesigned genetic code. Manipulation of iVTtRNA composition in the system enabled genetic code rewriting. Introduction of modified nucleotides into specific iVTtRNAs demonstrated to be effective for both protein yield and decoding fidelity, where the production yield of DHFR reached about 40% of the reaction with native tRNA at 30°C. The developed system will prove useful for studying decoding processes, and may be employed in genetic code and protein engineering applications.

    DOI PubMed

    Scopus

    39
    被引用数
    (Scopus)
  • In vitro reconstitution of functional small ribosomal subunit assembly for comprehensive analysis of ribosomal elements in E. coli

    Masaru Shimojo, Kazuaki Amikura, Keiko Masuda, Takashi Kanamori, Takuya Ueda, Yoshihiro Shimizu

    Communications Biology   3 ( 1 ) 142 - 142  2020年12月  [査読有り]

     概要を見る

    In vitro reconstitution is a powerful tool for investigating ribosome functions and biogenesis, as well as discovering new ribosomal features. In this study, we integrated all of the processes required for Escherichia coli small ribosomal subunit assembly. In our method, termed fully Recombinant-based integrated Synthesis, Assembly, and Translation (R-iSAT), assembly and evaluation of the small ribosomal subunits are coupled with ribosomal RNA (rRNA) synthesis in a reconstituted cell-free protein synthesis system. By changing the components of R-iSAT, including recombinant ribosomal protein composition, we coupled ribosomal assembly with ribosomal protein synthesis, enabling functional synthesis of ribosomal proteins and subsequent subunit assembly. In addition, we assembled and evaluated subunits with mutations in both rRNA and ribosomal proteins. The study demonstrated that our scheme provides new ways to comprehensively analyze any elements of the small ribosomal subunit, with the goal of improving our understanding of ribosomal biogenesis, function, and engineering.

    DOI PubMed

    Scopus

    24
    被引用数
    (Scopus)
  • Antitumor effects of ribavirin in combination with TMZ and IFN-β in malignant glioma cells

    Yushi Ochiai, Koichiro Sumi, Emiko Sano, Sodai Yoshimura, Shun Yamamuro, Akiyoshi Ogino, Takuya Ueda, Yutaka Suzuki, Tomohiro Nakayama, Hiroyuki Hara, Yoichi Katayama, Atsuo Yoshino

    Oncology Letters   20 ( 5 )  2020年11月

     概要を見る

    The prognosis of gioblastoma, the standard chemotherapy agent for which is temozolomide (TMZ), remains poor despite recent advances in multimodal treatments. Therefore, it is necessary to identify and develop novel therapeutics for this malignant disease. Ribavirin, an anti-viral agent which is one of the standard agents for treatment of chronic hepatitis C in combination with interferon (IFN), was recently revealed to have an antitumor potential towards various tumor cells, including malignant glioma cells. The aim of the present study was to examine the antitumor effect of ribavirin in combination with TMZ and IFN-β on glioma cells and to evaluate the possibility that such combinations might represent a novel candidate for glioblastoma therapy. The combination of ribavirin with TMZ and IFN-β displayed a significant cell growth inhibitory effect with a ribavirin dose-dependency, including a relatively low concentration of ribavirin, on not only TMZ-sensitive but also TMZ-resistant malignant glioma cells. The antitumor efficacy of such a combination further indicated a synergistic interaction when assessed by the Chou-Talalay method. Furthermore, flow cytometry analysis suggested that apoptosis induction was one of the possible biological processes underlying the synergistic antitumor effect of these triple combination treatments. Therefore, such combinations may be potentially important in the clinical setting for glioblastoma treatment, although further detailed studies, e.g. on the adverse effects, are required.

    DOI

    Scopus

    9
    被引用数
    (Scopus)
  • Antitumor effect of lenalidomide in malignant glioma cell lines

    Yuya Hanashima, Emiko Sano, Koichiro Sumi, Yoshinari Ozawa, Chihiro Yagi, Juri Tatsuoka, Sodai Yoshimura, Shun Yamamuro, Takuya Ueda, Tomohiro Nakayama, Hiroyuki Hara, Atsuo Yoshino

    Oncology Reports   43 ( 5 ) 1580 - 1590  2020年

     概要を見る

    Glioblastoma is a malignant brain tumor exhibiting highly aggressive proliferation and invasion capacities. Despite treatment by aggressive surgical resection and adjuvant therapy including temozolomide and radiation therapy, patient prognosis remains poor. Lenalidomide, a derivative of thalidomide, is known to be an immunomodulatory agent that has been used to treat hematopoietic malignancies. There are numerous studies revealing an antitumor effect of lenalidomide in hematopoietic cells, but not in glioma cells. The present study aimed to demonstrate the antitumor effect of lenalidomide on malignant glioma cell lines. The growth inhibition of malignant glioma cells (A-172, AM-38, T98G, U-138MG, U-251MG, and YH-13) by lenalidomide was assessed using a Coulter counter. The mechanism of the antitumor effect of lenalidomide was examined employing a fluorescence–activated cell sorter, western blot analysis, and quantitative real-time reverse transcriptional polymerase chain reaction (RT-qPCR) in malignant glioma cell lines (A-172, AM-38). The results revealed that the number of malignant glioma cells was decreased in a concentration-dependent manner by lenalidomide. DNA flow cytometric analysis demonstrated an increase in the ratio of cells at the G0/G1 phase following lenalidomide treatment. Western blot analysis and RT-qPCR revealed that p53 activation and the expression of p21 were increased in glioma cells treated with lenalidomide. Western blot analysis revealed that cleavage of PARP did not occur; however, increased expression of Bax protein, cleavage of caspase–9 and cleavage of caspase–3 were confirmed. Analysis by FACS also supported the conclusion that little apoptosis induction occurred following lenalidomide treatment of malignant glioma cell lines. In conclusion, lenalidomide exerts an antitumor effect on glioma cells due to alterations in cell cycle distribution.

    DOI PubMed

    Scopus

    8
    被引用数
    (Scopus)
  • The bacterial protein YidC accelerates MPIase-dependent integration of membrane proteins

    Masaru Sasaki, Hanako Nishikawa, Sonomi Suzuki, Michael Moser, Maria Huber, Katsuhiro Sawasato, Hideaki T. Matsubayashi, Kaoru Kumazaki, Tomoya Tsukazaki, Yutetsu Kuruma, Osamu Nureki, Takuya Ueda, Ken Ichi Nishiyama

    Journal of Biological Chemistry   294 ( 49 ) 18898 - 18908  2019年12月  [査読有り]

     概要を見る

    Bacterial membrane proteins are integrated into membranes through the concerted activities of a series of integration factors, including membrane protein integrase (MPIase). However, how MPIase activity is complemented by other integration factors during membrane protein integration is incompletely understood. Here, using inverted inner-membrane vesicle and reconstituted (proteo)liposome preparations from Escherichia coli cells, along with membrane protein integration assays and the PURE system to produce membrane proteins, we found that anti-MPIase IgG inhibits the integration of both the Sec-independent substrate 3L-Pf3 coat and the Sec-dependent substrate MtlA into E. coli membrane vesicles. MPIase-depleted membrane vesicles lacked both 3L-Pf3 coat and MtlA integration, indicating that MPIase is involved in the integration of both proteins. We developed a reconstitution system in which disordered spontaneous integration was precluded, which revealed that SecYEG, YidC, or both, are not sufficient for Sec-dependent and -independent integration. Although YidC had no effect on MPIase-dependent integration of Sec-independent substrates in the conventional assay system, YidC significantly accelerated the integration when the substrate amounts were increased in our PURE system- based assay. Similar acceleration by YidC was observed for MtlA integration. YidC mutants with amino acid substitutions in the hydrophilic cavity inside the membrane were defective in the acceleration of the Sec-independent integration. Of note, MPIase was up-regulated upon YidC depletion. These results indicate that YidC accelerates the MPIase-dependent integration of membrane proteins, suggesting that MPIase and YidC function sequentially and cooperatively during the catalytic cycle of membrane protein integration.

    DOI PubMed

    Scopus

    16
    被引用数
    (Scopus)
  • CdsA is involved in biosynthesis of glycolipid MPIase essential for membrane protein integration in vivo

    Katsuhiro Sawasato, Ryo Sato, Hanako Nishikawa, Naoki Iimura, Yuki Kamemoto, Kohki Fujikawa, Toshiyuki Yamaguchi, Yutetsu Kuruma, Yasushi Tamura, Toshiya Endo, Takuya Ueda, Keiko Shimamoto, Ken ichi Nishiyama

    Scientific Reports   9 ( 1 )  2019年12月  [査読有り]

     概要を見る

    MPIase is a glycolipid that is involved in membrane protein integration. Despite evaluation of its functions in vitro, the lack of information on MPIase biosynthesis hampered verification of its involvement in vivo. In this study, we found that depletion of CdsA, a CDP-diacylglycerol synthase, caused not only a defect in phospholipid biosynthesis but also MPIase depletion with accumulation of the precursors of both membrane protein M13 coat protein and secretory protein OmpA. Yeast Tam41p, a mitochondrial CDP-diacylglycerol synthase, suppressed the defect in phospholipid biosynthesis, but restored neither MPIase biosynthesis, precursor processing, nor cell growth, indicating that MPIase is essential for membrane protein integration and therefore for cell growth. Consistently, we observed a severe defect in protein integration into MPIase-depleted membrane vesicles in vitro. Thus, the function of MPIase as a factor involved in protein integration was proven in vivo as well as in vitro. Moreover, Cds1p, a eukaryotic CdsA homologue, showed a potential for MPIase biosynthesis. From these results, we speculate the presence of a eukaryotic MPIase homologue.

    DOI PubMed

    Scopus

    23
    被引用数
    (Scopus)
  • Artificial photosynthetic cell producing energy for protein synthesis

    Samuel Berhanu, Takuya Ueda, Yutetsu Kuruma

    Nature Communications   10 ( 1 )  2019年12月  [査読有り]

     概要を見る

    Attempts to construct an artificial cell have widened our understanding of living organisms. Many intracellular systems have been reconstructed by assembling molecules, however the mechanism to synthesize its own constituents by self-sufficient energy has to the best of our knowledge not been developed. Here, we combine a cell-free protein synthesis system and small proteoliposomes, which consist of purified ATP synthase and bacteriorhodopsin, inside a giant unilamellar vesicle to synthesize protein by the production of ATP by light. The photo-synthesized ATP is consumed as a substrate for transcription and as an energy for translation, eventually driving the synthesis of bacteriorhodopsin or constituent proteins of ATP synthase, the original essential components of the proteoliposome. The de novo photosynthesized bacteriorhodopsin and the parts of ATP synthase integrate into the artificial photosynthetic organelle and enhance its ATP photosynthetic activity through the positive feedback of the products. Our artificial photosynthetic cell system paves the way to construct an energetically independent artificial cell.

    DOI PubMed

    Scopus

    246
    被引用数
    (Scopus)
  • Promotion of TRAIL/Apo2L-induced apoptosis by low-dose interferon-β in human malignant melanoma cells

    Kazaana A, Sano E, Yoshimura S, Makita K, Hara H, Yoshino A, Ueda T

    Journal of Cellular Physiology   234 ( 8 ) 13510 - 13524  2019年08月  [査読有り]

    DOI PubMed

    Scopus

    12
    被引用数
    (Scopus)
  • Interferon-β sensitizes human malignant melanoma cells to temozolomide-induced apoptosis and autophagy

    Makita K, Hara H, Sano E, Okamoto Y, Ochiai Y, Harada T, Ueda T, Nakayama T, Aizawa S, Yoshino A

    International Journal of Oncology   54 ( 5 ) 1864 - 1874  2019年04月  [査読有り]

    DOI PubMed

    Scopus

    6
    被引用数
    (Scopus)
  • High-resolution crystal structure of peptidyl-tRNA hydrolase from Thermus thermophilus

    Ami Matsumoto, Yuji Uehara, Yoshihiro Shimizu, Takuya Ueda, Toshio Uchiumi, Kosuke Ito

    Proteins: Structure, Function and Bioinformatics   87 ( 3 ) 226 - 235  2019年03月  [査読有り]  [国際誌]

     概要を見る

    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the products of defective translation, to recycle the tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its activity is essential for bacterial viability. Here, we have determined the crystal structure of Pth from Thermus thermophilus (TtPth) at 1.00 Å resolution. This is the first structure of a Pth from a thermophilic bacterium and the highest resolution Pth structure reported so far. The present atomic resolution data enabled the calculation of anisotropic displacement parameters for all atoms, which revealed the directionality of the fluctuations of key regions for the substrate recognition. Comparisons between TtPth and mesophilic bacterial Pths revealed that their structures are similar overall. However, the structures of the N- and C-terminal, loop-helix α4, and helix α6 regions are different. In addition, the helix α1 to strand β4 region of TtPth is remarkably different from those of the mesophilic bacterial Pths, because this region is 9 or 10 amino acid residues shorter than those of the mesophilic bacterial Pths. This shortening seems to contribute to the thermostability of TtPth. To further understand the determinants for the thermostability of TtPth, we compared various structural factors of TtPth with those of mesophilic bacterial Pths. The data suggest that the decreases in accessible surface area and thermolabile amino acid residues, and the increases in ion pairs, hydrogen bonds, and proline residues cooperatively contribute to the thermostability of TtPth.

    DOI PubMed

    Scopus

    2
    被引用数
    (Scopus)
  • IFN‑β sensitizes TRAIL‑induced apoptosis by upregulation of death receptor 5 in malignant glioma cells

    Sodai Yoshimura, Emiko Sano, Yuya Hanashima, Shun Yamamuro, Koichiro Sumi, Takuya Ueda, Tomohiro Nakayama, Hiroyuki Hara, Atsuo Yoshino, Yoichi Katayama

    Oncology Reports   42 ( 6 ) 2635 - 2643  2019年

     概要を見る

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, induces apoptosis in cancer cells by binding to its receptors, death receptor 4 (DR4) and DR5, without affecting normal cells, and is therefore considered to be a promising antitumor agent for use in cancer treatment. However, several studies have indicated that most glioma cell lines display resistance to TRAIL-induced apoptosis. To overcome such resistance and to improve the efficacy of TRAIL-based therapies, identification of ideal agents for combinational treatment is important for achieving rational clinical treatment in glioblastoma patients. The main aim of this study was to investigate whether interferon-β (IFN-β) (with its pleiotropic antitumor activities) could sensitize malignant glioma cells to TRAIL-induced apoptosis using glioma cell lines. TRAIL exhibited a dose-dependent antitumor effect in all of the 7 types of malignant glioma cell lines, although the intensity of the effect varied among the cell lines. In addition, combined treatment with TRAIL (low clinical dose: 1 ng/ml) and IFN-β (clinically relevant concentration: 10 IU/ml) in A-172, AM-38, T98G, U-138MG and U-251MG demonstrated a more marked antitumor effect than TRAIL alone. Furthermore, the antitumor effect of the combined treatment with TRAIL and IFN-β may be enhanced via an extrinsic apoptotic system, and upregulation of DR5 was revealed to play an important role in this process in U‑138MG cells. These findings provide an experimental basis to suggest that combined treatment with TRAIL and IFN-β may offer a new therapeutic strategy for malignant gliomas.

    DOI PubMed

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    8
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    (Scopus)
  • Large-scale aggregation analysis of eukaryotic proteins reveals an involvement of intrinsically disordered regions in protein folding

    Eri Uemura, Tatsuya Niwa, Shintaro Minami, Kazuhiro Takemoto, Satoshi Fukuchi, Kodai Machida, Hiroaki Imataka, Takuya Ueda, Motonori Ota, Hideki Taguchi

    Scientific Reports   8 ( 1 )  2018年12月  [査読有り]

     概要を見る

    A subset of the proteome is prone to aggregate formation, which is prevented by chaperones in the cell. To investigate whether the basic principle underlying the aggregation process is common in prokaryotes and eukaryotes, we conducted a large-scale aggregation analysis of ~500 cytosolic budding yeast proteins using a chaperone-free reconstituted translation system, and compared the obtained data with that of ~3,000 Escherichia coli proteins reported previously. Although the physicochemical properties affecting the aggregation propensity were generally similar in yeast and E. coli proteins, the susceptibility of aggregation in yeast proteins were positively correlated with the presence of intrinsically disordered regions (IDRs). Notably, the aggregation propensity was not significantly changed by a removal of IDRs in model IDR-containing proteins, suggesting that the properties of ordered regions in these proteins are the dominant factors for aggregate formation. We also found that the proteins with longer IDRs were disfavored by E. coli chaperonin GroEL/ES, whereas both bacterial and yeast Hsp70/40 chaperones have a strong aggregation-prevention effect even for proteins possessing IDRs. These results imply that a key determinant to discriminate the eukaryotic proteomes from the prokaryotic proteomes in terms of protein folding would be the attachment of IDRs.

    DOI PubMed

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    18
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    (Scopus)
  • G-protein coupled receptor protein synthesis on a lipid bilayer using a reconstituted cell-free protein synthesis system

    Belay Gessesse, Takashi Nagaike, Koji Nagata, Yoshihiro Shimizu, Takuya Ueda

    Life   8 ( 4 )  2018年12月  [査読有り]  [国際誌]

     概要を見る

    Membrane proteins are important drug targets which play a pivotal role in various cellular activities. However, unlike cytosolic proteins, most of them are difficult-to-express proteins. In this study, to synthesize and produce sufficient quantities of membrane proteins for functional and structural analysis, we used a bottom-up approach in a reconstituted cell-free synthesis system, the PURE system, supplemented with artificial lipid mimetics or micelles. Membrane proteins were synthesized by the cell-free system and integrated into lipid bilayers co-translationally. Membrane proteins such as the G-protein coupled receptors were expressed in the PURE system and a productivity ranging from 0.04 to 0.1 mg per mL of reaction was achieved with a correct secondary structure as predicted by circular dichroism spectrum. In addition, a ligand binding constant of 27.8 nM in lipid nanodisc and 39.4 nM in micelle was obtained by surface plasmon resonance and the membrane protein localization was confirmed by confocal microscopy in giant unilamellar vesicles. We found that our method is a promising approach to study the different classes of membrane proteins in their native-like artificial lipid bilayer environment for functional and structural studies.

    DOI PubMed

    Scopus

    19
    被引用数
    (Scopus)
  • STEM-27. THE ALTERATION OF IMMUNOSUPPRESSIVE FUNCTION IN GLIOBLASTOMA WITH UNDIFFERENTIATED TRANSFORMATION

    Shun Yamamuro, Yuya Hanashima, Sodai Yoshimura, Emiko Sano, Takuya Ueda, Atsuo Yoshino

    Neuro-Oncology   20 ( suppl_6 ) vi249 - vi249  2018年11月  [査読有り]

    DOI

  • Reconstitution of 30S ribosomal subunits in vitro using ribosome biogenesis factors

    Daichi Tamaru, Kazuaki Amikura, Yoshihiro Shimizu, Knud H. Nierhaus, Takuya Ueda

    RNA   24 ( 11 ) 1512 - 1519  2018年11月  [査読有り]

     概要を見る

    Reconstitution of ribosomes in vitro from individual ribosomal proteins provides a powerful tool for understanding the ribosome assembly process including the sequential incorporation of ribosomal proteins. However, conventional assembly methods require high-salt conditions for efficient ribosome assembly. In this study, we reconstituted 30S ribosomal subunits from individually purified ribosomal proteins in the presence of ribosome biogenesis factors. In this system, two GTPases (Era and YjeQ) facilitated assembly of a 30S subunit exhibiting poly(U)-directed polyphenylalanine synthesis and native protein synthesis under physiological conditions. This in vitro system permits a study of the assembly process and function of ribosome biogenesis factors, and it will facilitate the generation of ribosomes from DNA without using cells.

    DOI PubMed

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    20
    被引用数
    (Scopus)
  • Construction of integrated gene logic-chip

    Takeya Masubuchi, Masayuki Endo, Ryo Iizuka, Ayaka Iguchi, Dong Hyun Yoon, Tetsushi Sekiguchi, Hao Qi, Ryosuke Iinuma, Yuya Miyazono, Shuichi Shoji, Takashi Funatsu, Hiroshi Sugiyama, Yoshie Harada, Takuya Ueda, Hisashi Tadakuma

    Nature Nanotechnology   13 ( 10 ) 933 - 940  2018年10月  [査読有り]

     概要を見る

    © 2018, The Author(s). In synthetic biology, the control of gene expression requires a multistep processing of biological signals. The key steps are sensing the environment, computing information and outputting products1. To achieve such functions, the laborious, combinational networking of enzymes and substrate-genes is required, and to resolve problems, sophisticated design automation tools have been introduced2. However, the complexity of genetic circuits remains low because it is difficult to completely avoid crosstalk between the circuits. Here, we have made an orthogonal self-contained device by integrating an actuator and sensors onto a DNA origami-based nanochip that contains an enzyme, T7 RNA polymerase (RNAP) and multiple target-gene substrates. This gene nanochip orthogonally transcribes its own genes, and the nano-layout ability of DNA origami allows us to rationally design gene expression levels by controlling the intermolecular distances between the enzyme and the target genes. We further integrated reprogrammable logic gates so that the nanochip responds to water-in-oil droplets and computes their small RNA (miRNA) profiles, which demonstrates that the nanochip can function as a gene logic-chip. Our approach to component integration on a nanochip may provide a basis for large-scale, integrated genetic circuits.

    DOI PubMed

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    37
    被引用数
    (Scopus)
  • De Novo Synthesis of Basal Bacterial Cell Division Proteins FtsZ, FtsA, and ZipA Inside Giant Vesicles

    Takumi Furusato, Fumihiro Horie, Hideaki T. Matsubayashi, Kazuaki Amikura, Yutetsu Kuruma, Takuya Ueda

    ACS Synthetic Biology   7 ( 4 ) 953 - 961  2018年04月  [査読有り]

     概要を見る

    Cell division is the most dynamic event in the cell cycle. Recently, efforts have been made to reconstruct it using the individual component proteins to obtain a better understanding of the process of self-reproduction of cells. However, such reconstruction studies are frequently hampered by difficulties in preparing membrane-associated proteins. Here we demonstrate a de novo synthesis approach based on a cell-free translation system. Genes for fundamental cell division proteins, FtsZ, FtsA, and ZipA, were expressed inside the lipid compartment of giant vesicles (GVs). The synthesized proteins showed polymerization, membrane localization, and eventually membrane deformation. Notably, we found that this morphological change of the vesicle is forced by only FtsZ and ZipA, which form clusters on the membrane at the vesicle interior. Our cell-free approach provides a platform for studying protein dynamics associated with lipid membrane and paves the way to create a synthetic cell that undergoes self-reproduction.

    DOI PubMed

    Scopus

    58
    被引用数
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  • Efficacy of ribavirin against malignant glioma cell lines: Follow-up study

    Yushi Ochiai, Emiko Sano, Yutaka Okamoto, Sodai Yoshimura, Kotaro Makita, Shun Yamamuro, Takashi Ohta, Akiyoshi Ogino, Hisashi Tadakuma, Takuya Ueda, Tomohiro Nakayama, Hiroyuki Hara, Atsuo Yoshino, Yoichi Katayama

    Oncology Reports   39 ( 2 ) 537 - 544  2018年02月  [査読有り]

     概要を見る

    Ribavirin, a nucleic acid analog, has been employed as an antiviral agent against RNA and DNA viruses and has become the standard agent used for chronic hepatitis C in combination with interferon-α2a. Furthermore, the potential antitumor efficacy of ribavirin has attracted increasing interest. Recently, we demonstrated a dose-dependent antitumor effect of ribavirin for seven types of malignant glioma cell lines. However, the mechanism underlying the antitumor effect of ribavirin has not yet been fully elucidated. Therefore, the main aim of the present study was to provide further relevant data using two types of malignant glioma cell lines (U-87MG and U-138MG) with different expression of MGMT. Dotted accumulations of γH2AX were found in the nuclei and increased levels of ATM and phosphorylated ATM protein expression were also observed following ribavirin treatment (10 μM of ribavirin, clinical relevant concentration) in both the malignant glioma cells, indicating double-strand breaks as one possible mechanism underlying the antitumor effect of ribavirin. In addition, based on assessements using FACS, ribavirin treatment tended to increase the G0/G1 phase, with a time-lapse, indicating the induction of G0/G1-phase arrest. Furthermore, an increased phosphorylated p53 and p21 protein expression was confirmed in both glioma cells. Additionally, analysis by FACS indicated that apoptosis was induced following ribavirin treatment and caspase cascade, downstream of the p53 pathway, which indicated the activation of both exogenous and endogenous apoptosis in both malignant glioma cell lines. These findings may provide an experimental basis for the clinical treatment of glioblastomas with ribavirin.

    DOI

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    13
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  • DNAオリガミを用いた直行性を有する転写デバイスの合理的設計

    増渕 岳也, 遠藤 政幸, 飯塚 怜, 井口 彩香, Yoon Dong, 関口 哲志, Qi Hao, 飯沼 良介, 宮薗 侑也, 庄子 習一, 船津 高志, 杉山 弘, 原田 慶恵, 上田 卓也, 多田隈 尚史

    生命科学系学会合同年次大会   2017年度   [2P - 1323]  2017年12月

  • Production and characterization of genetically modified human IL-11 variants

    Emiko Sano, Toshiaki Takei, Takuya Ueda, Kouhei Tsumoto

    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS   1861 ( 2 ) 205 - 217  2017年02月  [査読有り]

     概要を見る

    Interleukin-11 (IL-11) has been expected as a drug on severe thrombocytopenia caused by myelo-suppressive chemotherapy. Whereas, development of IL-11 inhibitor is also expected for a treatment against IL-11 related cancer progression. Here, we will demonstrate the creation of various kinds of genetically modified hIL-11s. Modified vectors were constructed by introducing N- or O-glycosylation site on the region of hIL-11 that does not belong to the core a.-helical motif based on the predicted secondary structure. N-terminal (N: between 22 to 23 aa), the first loop (M1:70 to 71 aa), the second loop (M2:114-115 aa), the third loop (M3:160-161 aa) and C-terminal (C: 200- aa) were selected for modification. A large scale production system was established and the characteristics of modified hIL-11s were evaluated. The structure was analyzed by amino acid sequence and composition analysis and CD-spectra. Glycan was assessed by monosaccharide composition analysis. Growth promoting activity and biological stability were analyzed by proliferation of T1165 cells. N-terminal modified proteins were well glycosylated and produced. Growth activity of 3NN with NASNASNAS sequence on N-terminal was about tenfold higher than wild type (WT). Structural and biological stabilities of 3NN were also better than WT and residence time in mouse blood was longer than WT. M1 variants lacked growth activity though they are well glycosylated and secondary structure is very stable. Both of 3NN and OM1 with AAATPAPG on M1 associated with hIL-11R strongly. These results indicate N-terminal and M1 variants will be expected for practical use as potent agonists or antagonists of hIL-11. (C) 2016 Elsevier B.V. All rights reserved.

    DOI

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    3
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    (Scopus)
  • Composition for synthesizing protein with reduced lipopolysaccharide contamination, method for producing protein using said composition

    Takuya Ueda

    Official Gazette of the United States Patent and Trademark Office Patents    2017年

  • Cell-free translation system: Development in biochemistry and advance in synthetic biology

    Kanamori, Takashi, Nagaike, Takashi, Kuruma, Yutetsu, Ueda, Takuya, Corporation, Genefrontier, Plaza, Todai-kashiwa Venture

    Journal of Japanese Biochemical Society   89 ( 2 ) 211 - 220  2017年  [査読有り]

    DOI

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    2
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  • The Termination Phase in Protein Synthesis is not Obligatorily Followed by the RRF/EF-G-Dependent Recycling Phase

    Bo Qin, Hiroshi Yamamoto, Takuya Ueda, Umesh Varshney, Knud H. Nierhaus

    JOURNAL OF MOLECULAR BIOLOGY   428 ( 18 ) 3577 - 3587  2016年09月  [査読有り]

     概要を見る

    It is general wisdom that termination of bacterial protein synthesis is obligatorily followed by recycling governed by the factors ribosomal recycling factor (RRF), EF-G, and IF3, where the ribosome dissociates into its subunits. In contrast, a recently described 70S-scanning mode of initiation holds that after termination, scanning of 70S can be triggered by fMet-tRNA to the initiation site of a downstream cistron. Here, we analyze the apparent conflict. We constructed a bicistronic mRNA coding for luciferases and showed with a highly resolved in vitro system that the expression of the second cistron did not at all depend on the presence of active RRF. An in vivo analysis cannot be performed in a straightforward way, since RRF is essential for viability and therefore, the RRF gene cannot be knocked out. However, we found an experimental window, where the RRF amount could be reduced to below 2.5%, and in this situation, the expression of the second cistron of a bicistronic luciferase mRNA was only moderately reduced. Both in vitro and in vivo results suggested that RRF-dependent recycling is not an obligatory step after termination, in agreement with the previous findings concerning 70S-scanning initiation. In this view, recycling after termination is a special case of the general RRF function, which happens whenever fMet-tRNA is not available for triggering 70S scanning. (C) 2016 Elsevier Ltd. All rights reserved.

    DOI

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    9
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    (Scopus)
  • The PURE system for the cell-free synthesis of membrane proteins (vol 10, pg 1328, 2015)

    Yutetsu Kuruma, Takuya Ueda

    NATURE PROTOCOLS   11 ( 3 ) 616 - 616  2016年03月  [査読有り]

    DOI

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    1
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  • Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp PCC 6803

    Rayakorn Yutthanasirikul, Takanori Nagano, Haruhiko Jimbo, Yukako Hihara, Takashi Kanamori, Takuya Ueda, Takamitsu Haruyama, Hiroki Konno, Keisuke Yoshida, Toru Hisabori, Yoshitaka Nishiyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   291 ( 11 ) 5860 - 5870  2016年03月  [査読有り]

     概要を見る

    Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

    DOI

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    33
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  • 70S-scanning initiation is a novel and frequent initiation mode of ribosomal translation in bacteria

    Hiroshi Yamamoto, Daniela Wittek, Romi Gupta, Bo Qin, Takuya Ueda, Roland Krause, Kaori Yamamoto, Renate Albrecht, Markus Pech, Knud H. Nierhaus

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   113 ( 9 ) E1180 - E1189  2016年03月  [査読有り]

     概要を見る

    According to the standard model of bacterial translation initiation, the small ribosomal 30S subunit binds to the initiation site of an mRNA with the help of three initiation factors (IF1-IF3). Here, we describe a novel type of initiation termed "70S-scanning initiation," where the 70S ribosome does not necessarily dissociate after translation of a cistron, but rather scans to the initiation site of the downstream cistron. We detailed the mechanism of 70S-scanning initiation by designing unique monocistronic and polycistronic mRNAs harboring translation reporters, and by reconstituting systems to characterize each distinct mode of initiation. Results show that 70S scanning is triggered by fMet-tRNA and does not require energy; the Shine-Dalgarno sequence is an essential recognition element of the initiation site. IF1 and IF3 requirements for the various initiation modes were assessed by the formation of productive initiation complexes leading to synthesis of active proteins. IF3 is essential and IF1 is highly stimulating for the 70S-scanning mode. The task of IF1 appears to be the prevention of untimely interference by ternary aminoacyl (aa)-tRNA center dot elongation factor thermo unstable (EF-Tu)center dot GTP complexes. Evidence indicates that at least 50% of bacterial initiation events use the 70S-scanning mode, underscoring the relative importance of this translation initiation mechanism.

    DOI

    Scopus

    68
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  • RNA Study Using DNA Nanotechnology

    Hisashi Tadakuma, Takeya Masubuchi, Takuya Ueda

    NANOTECHNOLOGY TOOLS FOR THE STUDY OF RNA   139   121 - 163  2016年  [査読有り]

     概要を見る

    Transcription is one of the fundamental steps of gene expression, where RNA polymerases (RNAPs) bind to their template genes and make RNAs. In addition to RNAP and the template gene, many molecules such as transcription factors are involved. The interaction and the effect of these factors depend on the geometry. Molecular layout of these factors, RNAP and gene is thus important. DNA nanotechnology is a promising technology that allows controlling of the molecular layout in the range of nanometer to micrometer scale with nanometer resolution; thus, it is expected to expand the RNA study beyond the current limit.

    DOI

    Scopus

  • DNA折り紙上の異なった方式による遺伝子転写ナノデバイスの理論的な設計(Rational design of orthogonal gene transcription nano device on DNA origami)

    増渕 岳也, 多田隈 向史, 飯塚 怜, 遠藤 政幸, 船津 高志, 杉山 弘, 原田 慶恵, 上田 卓也

    日本生化学会大会・日本分子生物学会年会合同大会講演要旨集   88回・38回   [3LBA089] - [3LBA089]  2015年12月

  • Comprehensive study of liposome-assisted synthesis of membrane proteins using a reconstituted cell-free translation system

    Tatsuya Niwa, Yoshihiro Sasaki, Eri Uemura, Shugo Nakamura, Minato Akiyama, Mitsuru Ando, Shinichi Sawada, Sada-atu Mukai, Takuya Ueda, Hideki Taguchi, Kazunari Akiyoshi

    SCIENTIFIC REPORTS   5  2015年12月  [査読有り]

     概要を見る

    Membrane proteins play pivotal roles in cellular processes and are key targets for drug discovery. However, the reliable synthesis and folding of membrane proteins are significant problems that need to be addressed owing to their extremely high hydrophobic properties, which promote irreversible aggregation in hydrophilic conditions. Previous reports have suggested that protein aggregation could be prevented by including exogenous liposomes in cell-free translation processes. Systematic studies that identify which membrane proteins can be rescued from irreversible aggregation during translation by liposomes would be valuable in terms of understanding the effects of liposomes and developing applications for membrane protein engineering in the context of pharmaceutical science and nanodevice development. Therefore, we performed a comprehensive study to evaluate the effects of liposomes on 85 aggregation-prone membrane proteins from Escherichia coli by using a reconstituted, chemically defined cell-free translation system. Statistical analyses revealed that the presence of liposomes increased the solubility of > 90% of the studied membrane proteins, and ultimately improved the yields of the synthesized proteins. Bioinformatics analyses revealed significant correlations between the liposome effect and the physicochemical properties of the membrane proteins.

    DOI

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    31
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  • Antitumorigenic effect of interferon-beta by inhibition of undifferentiated glioblastoma cells

    Shun Yamamuro, Emiko Sano, Yutaka Okamoto, Yushi Ochiai, Takashi Ohta, Akiyoshi Ogino, Atsushi Natsume, Toshihiko Wakabayashi, Takuya Ueda, Hiroyuki Hara, Tomohiro Nakayama, Atsuo Yoshino, Yoichi Katayama

    INTERNATIONAL JOURNAL OF ONCOLOGY   47 ( 5 ) 1647 - 1654  2015年11月  [査読有り]

     概要を見る

    Glioma stem-like cells (GSCs) are undifferentiated cells that are considered to be an origin of glioblastomas. Furthermore, they may contribute to treatment resistance and recurrence in glioblastomas. GSCs differentiate into differentiated glioma cells (non-glioma stem-like cells: non-GSCs), and interconversion might occur between GSCs and non-GSCs. We investigated whether interferon-beta (IFN-beta) could exert any efficacy towards GSCs or such interconversion processes. The neural stem cell marker CD133 and pluripotency marker Nanog in GSCs were analyzed to evaluate their differentiation levels. GSCs were considered to undergo differentiation into non-GSCs upon serum exposure, since the expression of CD133 and Nanog in the GSCs was negatively affected. Furthermore, the cells regained their undifferentiated features upon removal of the serum. However, we verified that IFN-beta reduced cell proliferation and tumor sphere formation in GSCs, and induced suppression of the restoration of such undifferentiated features. In addition, we also confirmed that IFN-beta suppressed the acquisition process of undifferentiated features in human malignant glioma cell lines. Our data thus suggest that IFN-beta could be an effective agent not only through its cell growth inhibitory effect on GSCs but also as a means of targeting the interconversion between GSCs and non-GSCs, indicating the possibility of IFN-beta being used to prevent treatment resistance and recurrence in glioblastomas, via the inhibition of undifferentiated features.

    DOI

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    6
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  • Large-scale analysis of macromolecular crowding effects on protein aggregation using a reconstituted cell-free translation system

    Tatsuya Niwa, Ryota Sugimoto, Lisa Watanabe, Shugo Nakamura, Takuya Ueda, Hideki Taguchi

    FRONTIERS IN MICROBIOLOGY   6 ( OCT )  2015年10月  [査読有り]

     概要を見る

    Proteins must fold into their native structures in the crowded cellular environment, to perform their functions. Although such macromolecular crowding has been considered to affect the folding properties of proteins, large-scale experimental data have so far been lacking. Here, we individually translated 142 Escherichia colt cytoplasmic proteins using a reconstituted cell-free translation system in the presence of macromolecular crowding reagents (MCRs), Ficoll 70 or dextran 70, and evaluated the aggregation propensities of 142 proteins. The results showed that the MGR effects varied depending on the proteins, although the degree of these effects was modest. Statistical analyses suggested that structural parameters were involved in the effects of the MCRs. Our dataset provides a valuable resource to understand protein folding and aggregation inside cells.

    DOI

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    12
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  • The PURE system for the cell-free synthesis of membrane proteins

    Yutetsu Kuruma, Takuya Ueda

    NATURE PROTOCOLS   10 ( 9 ) 1328 - 1344  2015年09月  [査読有り]

     概要を見る

    Cell-free gene expression systems are biotechnological tools for the in vitro production of proteins of interest. The addition of membrane vesicles (liposomes) enables the production of membrane proteins, including those in large-molecular-weight complexes, such as the SecYEG translocon or ATP synthase. Here we describe a protocol for the cell-free synthesis of membrane proteins using the protein synthesis using recombinant elements (PURE) system, and for subsequent quantification of products and analyses of membrane localization efficiency, product orientation in the membrane and complex formation in the membrane. In addition, measurements of ATP synthase activity are used as an example to demonstrate the functional nature of the cell-free synthesized proteins. This protocol allows the rapid production and the detailed analysis of membrane proteins, and the complete process from template DNA preparation to activity measurement can be accomplished within 1 d. In contrast to alternative methods using living cells, this protocol can also help to prevent the difficulties in membrane protein purification and the risks of protein aggregation during reconstitution into lipid membranes.

    DOI

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    101
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  • Oxidation of translation factor EF-G transiently retards the translational elongation cycle in Escherichia coli

    Takanori Nagano, Rayakorn Yutthanasirikul, Yukako Hihara, Toru Hisabori, Takashi Kanamori, Nono Takeuchi, Takuya Ueda, Yoshitaka Nishiyama

    JOURNAL OF BIOCHEMISTRY   158 ( 2 ) 165 - 172  2015年08月  [査読有り]

     概要を見る

    In Escherichia coli, elongation factor G (EF-G), a key protein in translational elongation, is particularly susceptible to oxidation. We demonstrated previously that EF-G is inactivated upon formation of an intramolecular disulphide bond. However, the details of the mechanism by which the oxidation of EF-G inhibits the function of EF-G on the ribosome remain to be elucidated. When we oxidized EF-G with hydrogen peroxide, neither the insertion of EF-G into the ribosome nor single-cycle translocation activity in vitro was affected. However, the GTPase activity and the dissociation of EF-G from the ribosome were suppressed when EF-G was oxidized. The synthesis of longer peptides was suppressed to a greater extent than that of a shorter peptide when EF-G was oxidized. Thus, the formation of the disulphide bond in EF-G might interfere with the hydrolysis of GTP that is coupled with dissociation of EF-G from the ribosome and might thereby retard the turnover of EF-G within the translational machinery. When we added thioredoxin to the suppressed translation system that included oxidized EF-G, translational activity was almost immediately restored. We propose that oxidation of EF-G might provide a regulatory mechanism for transient and reversible suppression of translation in E. coli under oxidative stress.

    DOI

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    11
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  • Single-Molecule Analysis of the Target Cleavage Reaction by the Drosophila RNAi Enzyme Complex

    Chunyan Yao, Hiroshi M. Sasaki, Takuya Ueda, Yukihide Tomari, Hisashi Tadakuma

    MOLECULAR CELL   59 ( 1 ) 125 - 132  2015年07月  [査読有り]

     概要を見る

    Small interfering RNAs (siRNAs) direct cleavage of complementary target RNAs via an RNA-induced silencing complex (RISC) that contains Argonatute2 protein at its core. However, what happens after target cleavage remains unclear. Here we analyzed the cleavage reaction by Drosophila Argonaute2-RISC using single-molecule imaging and revealed a series of intermediate states in target recognition, cleavage, and product release. Our data suggest that, after cleavage, RISC generally releases the 50 cleavage fragment from the guide 30 supplementary region first and then the 30 fragment from the seed region, highlighting the reinforcement of the seed pairing in RISC. However, this order can be reversed by extreme stabilization of the 30 supplementary region or mismatches in the seed region. Therefore, the release order of the two cleavage fragments is influenced by the stability in each region, in contrast to the unidirectional base pairing propagation from the seed to the 30 supplementary region upon target recognition.

    DOI

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    40
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  • Characterization of glioma stem-like cells from human glioblastomas

    Shun Yamamuro, Yutaka Okamoto, Emiko Sano, Yushi Ochiai, Akiyoshi Ogino, Takashi Ohta, Hiroyuki Hara, Takuya Ueda, Tomohiro Nakayama, Atsuo Yoshino, Yoichi Katayama

    INTERNATIONAL JOURNAL OF ONCOLOGY   47 ( 1 ) 91 - 96  2015年07月  [査読有り]

     概要を見る

    Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs.

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    21
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  • PH responsiveness of fibrous assemblies of repeat-sequence amphipathic α-helix polypeptides

    Takei T, Tsumoto K, Okonogi A, Kimura A, Kojima S, Yazaki K, Takei T, Ueda T, Miura K.-I

    Protein Science   24 ( 5 ) 883 - 894  2015年05月  [査読有り]

    DOI

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    3
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  • Differential Effects of IFN-β on the Survival and Growth of Human Vascular Smooth Muscle and Endothelial Cells

    Emiko Sano, Shinya Tashiro, Kouhei Tsumoto, Takuya Ueda

    BioResearch Open Access   4 ( 1 ) 1 - 15  2015年01月  [査読有り]

     概要を見る

    It has been documented that interferon (IFN)-β is effective against the genesis of atherosclerosis or hyperplastic arterial disease in animal model. The main mechanism of the efficacy was antiproliferative action on the growth of vascular smooth muscle cells (SMC). To understand more about the mechanisms that are responsible for the efficacy, we examined minutely the effects of IFN-β on the apoptosis and growth of vascular SMC and endothelial cells (EC). IFN-β enhanced SMC apoptosis in serum starved medium. Conversely, EC apoptosis induced by serum and growth factor deprivation was inhibited by IFN-β. The induction of SMC apoptosis and anti-apoptotic effect on EC linked to the expression of pro-apoptotic bax mRNA and caspase-3 activities. Anti-apoptotic bcl-2 mRNA was also up-regulated in EC. IFN-β inhibited SMC growth in a dose dependent manner. However, the growth of EC was rather enhanced by a low dose of IFNs. The antiproliferative effect on SMC associated with the activation of p21 and increase of G0/G1 arrested cells. The growth stimulation on EC was considered to link with increase of S and G2/M phase cells. SMC produced IFN-β in response to various stimulants. However, IFN-β was not induced in EC. These suggested that endogenous IFN-β from SMC may act on EC and affect to EC functions. In this study, it was clarified that IFN-β enhances SMC apoptosis and inhibits the EC apoptosis, and stimulates the EC growth. These effects were considered to contribute to a cure against hyperplastic arterial diseases as the mechanisms in the efficacy of IFN-β.

    DOI

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    5
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    (Scopus)
  • Efficacy of ribavirin against malignant glioma cell lines

    Akiyoshi Ogino, Emiko Sano, Yushi Ochiai, Shun Yamamuro, Shinya Tashiro, Kazunari Yachi, Takashi Ohta, Takao Fukushima, Yutaka Okamoto, Kouhei Tsumoto, Takuya Ueda, Atsuo Yoshino, Yoichi Katayama

    ONCOLOGY LETTERS   8 ( 6 ) 2469 - 2474  2014年12月  [査読有り]

     概要を見る

    Ribavirin (1-beta-D-ribofuranosy-1,2,4-triazole-3-carboxamide) has been widely administered as an antiviral agent against RNA and DNA viruses. Ribavirin, in combination with interferon, has predominantly been applied in the treatment of the hepatitis C virus infection and its potential antitumor efficacy has recently become a point of interest. The aim of the present study was to evaluate the effect of ribavirin on the growth of malignant glioma cells, to identify novel predictive genes in malignant glioma cells (by analyzing gene expression profiles) and to assess the influence of ribavirin on the cell cycle of malignant glioma cells. The present study evaluated the antitumor efficacy of ribavirin against various malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13). After culturing the cells in ribavirin-containing culture medium (final concentration, 0-1,000 mu M) for 72 h, the viable proliferated cells were harvested and counted. The half maximal inhibitory concentration of ribavirin, with regard to the growth of the malignant glioma cell lines, was determined from the concentration of ribavirin required for 50% growth inhibition in comparison to the untreated control cells. Furthermore, the current study identified the genes in which the gene expression levels correlated with the ribavirin sensitivity of the malignant glioma cells lines, using a high-density oligonucleotide array. Finally, cell cycle analysis was performed on the U-87MG cell line. It was identified that ribavirin inhibited the growth of all of the malignant glioma cell lines in a dose-dependent manner, although the ribavirin sensitivity varied between each cell line. Of the extracted genes, PDGFRA demonstrated the strongest positive correlation between gene expression level and ribavirin sensitivity. Cell cycle analysis of the U-87MG cell line demonstrated that ribavirin treatment induces G0/G1 arrest and thus may be an effective agent for inhibiting malignant glioma cell growth. Therefore, the results of the current study indicate that ribavirin may have potential as a therapeutic agent in the treatment of malignant gliomas.

    DOI

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    15
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  • Cell-Free Synthesis of SecYEG Translocon as the Fundamental Protein Transport Machinery

    Hideaki Matsubayashi, Yutetsu Kuruma, Takuya Ueda

    ORIGINS OF LIFE AND EVOLUTION OF BIOSPHERES   44 ( 4 ) 331 - 334  2014年12月  [査読有り]

     概要を見る

    The cell membrane has many indispensable functions for sustaining cell alive besides a role as merely outer envelope. The most of such functions are implemented by membrane embedded proteins that are emerged through the membrane integration machinery, SecYEG translocon. Here, we synthesized SecYEG by expressing the corresponding gene in vitro to study the process of functionalization of the cell membrane.

    DOI

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    9
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  • PURE ribosome display and its application in antibody technology

    Takashi Kanamori, Yasuhiro Fujino, Takuya Ueda

    BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS   1844 ( 11 ) 1925 - 1932  2014年11月  [査読有り]

     概要を見る

    Ribosome display utilizes formation of the mRNA-ribosome-polypeptide ternary complex in a cell-free protein synthesis system to link genotype (mRNA) to phenotype (polypeptide). However, the presence of intrinsic components, such as nucleases in the cell-extract-based cell-free protein synthesis system, reduces the stability of the ternary complex, which would prevent attainment of reliable results. We have developed an efficient and highly controllable ribosome display system using the PURE (Protein synthesis Using Recombinant Elements) system. The mRNA-ribosome-polypeptide ternary complex is highly stable in the PURE system, and the selected mRNA can be easily recovered because activities of nucleases and other inhibitory factors are very low in the PURE system. We have applied the PURE ribosome display to antibody engineering approaches, such as epitope mapping and affinity maturation of antibodies, and obtained results showing that the PURE ribosome display is more efficient than the conventional method. We believe that the PURE ribosome display can contribute to the development of useful antibodies. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody. (C) 2014 Elsevier B.V. All rights reserved.

    DOI

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    23
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  • Purified cell-free systems as standard parts for synthetic biology

    Hideaki Matsubayashi, Takuya Ueda

    CURRENT OPINION IN CHEMICAL BIOLOGY   22   158 - 162  2014年10月  [査読有り]

     概要を見る

    A cell free protein synthesis approach is extensively used for biochemical and synthetic biology researches. Unlike lysate based cell free systems, the PURE system is reconstituted with individually purified factors essential for transcriptional and translational processes. Hence, the components in the PURE system can be definitely manipulated as per the desired situation. Because of this high controllability, the PURE system has been applied to a wide range of research scene, such as biochemical analysis in reconstructed system, in vitro protein engineering, reconstitution of an artificial cell. We believe that this purified cell-free protein synthesis system become a basal technology to advance synthetic biology.

    DOI

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    30
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    (Scopus)
  • Transfer RNA Synthesis and Regulation

    Hiroyuki Hori, Chie Tomikawa, Akira Hirata, Yukimatsu Toh, Kozo Tomita, Takuya Ueda, Kimitsuna Watanabe

    eLS    2014年09月

    DOI

  • Ribosome Rescue and Translation Termination at Non-Standard Stop Codons by ICT1 in Mammalian Mitochondria

    Shiori Akabane, Takuya Ueda, Knud H. Nierhaus, Nono Takeuchi

    PLOS GENETICS   10 ( 9 ) e1004616  2014年09月  [査読有り]

     概要を見る

    Release factors (RFs) govern the termination phase of protein synthesis. Human mitochondria harbor four different members of the class 1 RF family: RF1Lmt/mtRF1a, RF1mt, C12orf65 and ICT1. The homolog of the essential ICT1 factor is widely distributed in bacteria and organelles and has the peculiar feature in human mitochondria to be part of the ribosome as a ribosomal protein of the large subunit. The factor has been suggested to rescue stalled ribosomes in a codonindependent manner. The mechanism of action of this factor was obscure and is addressed here. Using a homologous mitochondria system of purified components, we demonstrate that the integrated ICT1 has no rescue activity. Rather, purified ICT1 binds stoichiometrically to mitochondrial ribosomes in addition to the integrated copy and functions as a general rescue factor, i.e. it releases the polypeptide from the peptidyl tRNA from ribosomes stalled at the end or in the middle of an mRNA or even from non-programmed ribosomes. The data suggest that the unusual termination at a sense codon (AGA/G) of the oxidative-phosphorylation enzymes CO1 and ND6 is also performed by ICT1 challenging a previous model, according to which RF1Lmt/mtRF1a is responsible for the translation termination at non-standard stop codons. We also demonstrate by mutational analyses that the unique insertion sequence present in the N-terminal domain of ICT1 is essential for peptide release rather than for ribosome binding. The function of RF1mt, another member of the class1 RFs in mammalian mitochondria, was also examined and is discussed.

    DOI

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    55
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  • Protein synthesis yield increased 72 times in the cell-free PURE system

    Kirsten Jackson, Takashi Kanamori, Takuya Ueda, Z. Hugh Fan

    INTEGRATIVE BIOLOGY   6 ( 8 ) 781 - 788  2014年08月  [査読有り]

     概要を見る

    Compared to cell-based protein expression, cell-free protein synthesis (CFPS) offers several advantages including a greater control over system additives. This control is further enhanced with a CFPS system called the Protein synthesis Using Recombinant Elements (PURE) system, which consists of 108 purified transcriptional and translational elements. With the PURE system, all elements are known, nuclease and protease activities are reduced, and the concentration of each element can be optimized for maximal protein expression. However, protein expression yield with this system is relatively low due to the consumption of nutrients and energy molecules as well as the accumulation of inhibitory byproducts in the batch format. To enhance protein expression with the PURE system, we developed a feeding solution that was optimized using a miniaturized fluid array device (mu FAD) in a continuous-exchange cell-free (CECF) format. The device enabled (1) continuous supply of energy/nutrient molecules from the feeding solution to the reaction solution where protein synthesis occurred, and (2) simultaneous removal of inhibitory expression byproducts from the reaction solution to the feeding solution. Consequently, the synthesis yield of green fluorescent protein (GFP) increased 72.5-fold in comparison with the same reaction in the conventional batch format.

    DOI

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    17
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  • In Vitro Synthesis of the E. coli Sec Translocon from DNA

    Hideaki Matsubayashi, Yutetsu Kuruma, Takuya Ueda

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   53 ( 29 ) 7535 - 7538  2014年07月  [査読有り]

     概要を見る

    Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane-related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single- and multi-span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.

    DOI

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    65
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  • Role of Positions e and g in the Fibrous Assembly Formation of an Amphipathic alpha-Helix-Forming Polypeptide

    Toshiaki Takei, Kouhei Tsumoto, Masakuni Yoshino, Shuichi Kojima, Kazumori Yazaki, Takuya Ueda, Tsunetomo Takei, Fumio Arisaka, Kin-ichiro Miura

    BIOPOLYMERS   102 ( 3 ) 260 - 272  2014年05月  [査読有り]

     概要を見る

    We previously characterized alpha 3, a polypeptide that has a three times repeated sequence of seven amino acids (abc-defg: LETLAKA) and forms fibrous assemblies composed of amphipathic alpha-helices. Upon comparison of the amino acid sequences of alpha 3 with other alpha-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven alpha 3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The alpha-helix forming abilities of the substituted polypeptides were less than that of alpha 3. The polypeptides with amino acid substitutions at position g and the polypeptide KE alpha 3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed beta-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies. (C) 2014 Wiley Periodicals, Inc.

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    3
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  • Signal Recognition Particle and SecA Cooperate during Export of Secretory Proteins with Highly Hydrophobic Signal Sequences

    Yufan Zhou, Takuya Ueda, Matthias Mueller

    PLOS ONE   9 ( 4 )  2014年04月  [査読有り]

     概要を見る

    The Sec translocon of bacterial plasma membranes mediates the linear translocation of secretory proteins as well as the lateral integration of membrane proteins. Integration of many membrane proteins occurs co-translationally via the signal recognition particle (SRP)-dependent targeting of ribosome-associated nascent chains to the Sec translocon. In contrast, translocation of classical secretory proteins across the Sec translocon is a post-translational event requiring no SRP but the motor protein SecA. Secretory proteins were, however, reported to utilize SRP in addition to SecA, if the hydrophobicity of their signal sequences exceeds a certain threshold value. Here we have analyzed transport of this subgroup of secretory proteins across the Sec translocon employing an entirely defined in vitro system. We thus found SecA to be both necessary and sufficient for translocation of secretory proteins with hydrophobic signal sequences, whereas SRP and its receptor improved translocation efficiency. This SRP-mediated boost of translocation is likely due to the early capture of the hydrophobic signal sequence by SRP as revealed by site-specific photo cross-linking of ribosome nascent chain complexes.

    DOI

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    10
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  • Substrate Specificity of tRNA (Adenine-1-)-methyltransferase from Thermus thermophilus HB27

    Takuya Ueda

    Bioscience, Biotechnology, and Biochemistry    2014年

    DOI

  • In Vitro Reconstruction of Functional Membrane

    Takuya Ueda

    Alife: the Fourteenth International Conference on the Synthesis and Simulation of Living Systems    2014年

    DOI

  • 1P316 Rational design of orthogonal gene transcription nano device on DNA origami(28. Bioengineering,Poster,The 52nd Annual Meeting of the Biophysical Society of Japan(BSJ2014))

    Masubuchi Takeya, Tadakuma Hisashi, Endo Masayuki, Sugiyama Hiroshi, Harada Yoshie, Ueda Takuya

    生物物理   54 ( 1 ) S193  2014年

    DOI CiNii

  • The PURE system for protein production

    Yoshihiro Shimizu, Yutetsu Kuruma, Takashi Kanamori, Takuya Ueda

    Methods in Molecular Biology   1118   275 - 284  2014年  [査読有り]

     概要を見る

    In the field of molecular biology or biochemistry, preparation and use of purified proteins involved in a certain biological system is crucial for understanding their mechanisms and functions in cells or organisms. The recent progress in a cell-free translation system allows us to prepare proteins in a test tube directly from cDNAs that encode the amino acid sequences. The use of the reconstituted cell-free translation system termed PURE (Protein synthesis Using Recombinant Elements) for these purposes is effective in several applications. Here we describe methods of recombinant protein expression using the PURE system for molecular biological or biochemical studies. © 2014 Springer Science+Business Media, LLC.

    DOI PubMed

    Scopus

    29
    被引用数
    (Scopus)
  • Cell-free protein synthesis system

    Matsubayashi, H., Ueda, T.

    Kobunshi   63 ( 6 ) 379 - 381  2014年  [査読有り]

  • 大腸菌全タンパク質の凝集性とシャペロン効果の網羅的な解析

    丹羽 達也, 上田 卓也, 田中 英樹

    生物物理   53 ( 6 ) 309 - 312  2013年11月

     概要を見る

    Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. However, the relationship between the propensity to form aggregates and primary sequence and preferences of chaperones for substrates has not been understood. We comprehensively analyzed the aggregation propensity of all Escherichia coli proteins and the aggregation prevention effect of three major chaperones for aggregation-prone proteins by using a reconstituted chaperone-free translation system (PURE system). The resource obtained here can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects.<br>

    DOI CiNii

  • Unbiased Tracking of the Progression of mRNA and Protein Synthesis in Bulk and in Liposome-Confined Reactions

    Pauline van Nies, Zohreh Nourian, Maurits Kok, Roeland van Wijk, Jonne Moeskops, Ilja Westerlaken, Jos M. Poolman, Rienk Eelkema, Jan H. van Esch, Yutetsu Kuruma, Takuya Ueda, Christophe Danelon

    CHEMBIOCHEM   14 ( 15 ) 1963 - 1966  2013年10月  [査読有り]

     概要を見る

    The compartmentalization of a cell-free gene expression system inside a self-assembled lipid vesicle is envisioned as the simplest chassis for the construction of a minimal cell. Although crucial for its realization, quantitative understanding of the dynamics of gene expression in bulk and liposome-confined reactions is scarce. Here, we used two orthogonal fluorescence labeling tools to report the amounts of mRNA and protein produced in a reconstituted biosynthesis system, simultaneously and in real-time. The Spinach RNA aptamer and its fluorogenic probe were used for mRNA detection. Applying this dual-reporter assay to the analysis of transcript and protein production inside lipid vesicles revealed that their levels are uncorrelated, most probably a consequence of the low copy-number of some components in liposome-confined reactions. We believe that the stochastic nature of gene expression should be appreciated as a design principle for the assembly of a minimal cell.

    DOI

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    36
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  • Translation Enhancer Improves the Ribosome Liberation from Translation Initiation

    Shuntaro Takahashi, Hiroyuki Furusawa, Takuya Ueda, Yoshio Okahata

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   135 ( 35 ) 13096 - 13106  2013年09月  [査読有り]

     概要を見る

    For translation initiation in bacteria, the Shine-Dalgarno (SD) and anti-SD sequence of the 30S subunit play key roles for specific interactions between ribosomes and mRNAs to determine the exact position of the translation initiation region. However, ribosomes also must dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream of the SD sequences of mRNAs, which likely contribute to a direct interaction with ribosome protein Si, enhance the yields of protein biosynthesis. Nevertheless, the mechanism of the effect of translation enhancers to initiate the translation is still unknown. In this paper, we investigated the effects of the SD and enhancer sequences on the binding kinetics of the 30S ribosomal subunits to mRNAs and their translation efficiencies. mRNAs with both the SD and translation enhancers promoted the amount of protein synthesis but destabilized the interaction between the 30S subunit and mRNA by increasing the dissociation rate constant (k(off)) of the 30S subunit. Based on a model for kinetic parameters, a 16-fold translation efficiency could be achieved by introducing a tandem repeat of adenine sequences (A20) between the SD and translation enhancer sequences. Considering the results of this study, translation enhancers with an SD sequence regulate ribosomal liberation from translation initiation to determine the translation efficiency of the downstream coding region.

    DOI

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    30
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  • Nuclear Respiratory Factor 2 beta (NRF-2 beta) Recruits NRF-2 alpha to the Nucleus by Binding to Importin-alpha:beta via an Unusual Monopartite-Type Nuclear Localization Signal

    Rippei Hayashi, Nono Takeuchi, Takuya Ueda

    JOURNAL OF MOLECULAR BIOLOGY   425 ( 18 ) 3536 - 3548  2013年09月  [査読有り]

     概要を見る

    Nuclear respiratory factor 2 (NRF-2) is a mammalian transcription factor composed of two distinct and unrelated proteins: NRF-2 alpha, which binds to DNA through its Ets domain, and NRF-2 beta, which contains the transcription activation domain. The activity of NRF-2 in neurons is regulated by nuclear localization; however, the mechanism by which NRF-2 is imported into the nucleus remains unknown. By using in vitro nuclear import assays and immuno-cytofluorescence, we dissect the nuclear import pathways of NRF-2. We show that both NRF-2 alpha and NRF-2 beta contain intrinsic nuclear localization signals (NLSs): the Ets domain within NRF-2 alpha and the NLS within NRF-2 beta (amino acids 311/321: EEPPAKRQCIE) that is recognized by importin-alpha:beta. When NRF-2 alpha and NRF-2 beta form a complex, the nuclear import of NRF-2 alpha beta becomes strictly dependent on the NLS within NRF-2 beta. Therefore, the nuclear import mechanism of NRF-2 is unique among Ets factors. The NRF-2 beta NLS contains only two lysine/arginine residues, unlike other known importin-alpha:beta-dependent NLSs. Using ELISA-based binding assays, we show that it is bound by importin-a in almost the same manner and with similar affinity to that of the classical monopartite NLSs, such as c-myc and SV40 T-antigen NLSs. However, the part of the tryptophan array of importin-alpha that is essential for the recognition of classical monopartite NLSs by generating apolar pockets for the P-3 and the P-5 lysine/arginine side chains is not required for the recognition of the NRF-2 beta NLS. We conclude that the NRF-2 beta NLS is an unusual but is, nevertheless, a bona fide monopartite-type NLS. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

    DOI

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    3
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  • Direct Monitoring of Initiation Factor Dynamics through Formation of 30S and 70S TranslationInitiation Complexes on a Quartz Crystal Microbalance

    Shuntaro Takahashi, Hidemi Isobe, Takuya Ueda, Yoshio Okahata

    CHEMISTRY-A EUROPEAN JOURNAL   19 ( 21 ) 6807 - 6816  2013年05月  [査読有り]

     概要を見る

    Translation initiation is a dynamic and complicated process requiring the building a 70S initiation complex (70S-IC) composed of a ribosome, mRNA, and an initiator tRNA. During the formation of the 70S-IC, initiation factors (IFs: IF1, IF2, and IF3) interact with a ribosome to form a 30S initiation complex (30S-IC) and a 70S-IC. Although some spectroscopic analyses have been performed, the mechanism of binding and dissociation of IFs remains unclear. Here, we employed a 27MHz quartz crystal microbalance (QCM) to evaluate the process of bacterial IC formation in translation initiation by following frequency changes (mass changes). IFs (IF1, IF2, and IF3), N-terminally fused to biotin carboxyl carrier protein (bio-BCCP), were immobilized on a Neutravidin-covered QCM plate. By using bio-BCCP-IF2 immobilized to the QCM, three steps of the formation of ribosomal initiation complex could be sequentially observed as simple mass changes in real time: binding of a 30S complex to the immobilized IF2, a recruitment of 50S to the 30S-IC, and formation of the 70S-IC. The kinetic parameters implied that the release of IF2 from the 70S-IC could be the rate-limiting step in translation initiation. The IF3-immobilized QCM revealed that the affinity of IF3 for the 30S complex decreased upon the addition of mRNA and fMet-tRNAfMet but did not lead to complete dissociation from the 30S-IC. These results suggest that IF3 binds and stays bound to ICs, and its interaction mode is altered during the formation of 30S-IC and 70S-IC and is finally induced to dissociate from ICs by 50S binding. This methodology demonstrated here is applicable to investigate the role of IFs in translation initiation driven by other pathways.

    DOI

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    5
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  • Human G-proteins, ObgH1 and Mtg1, associate with the large mitochondrial ribosome subunit and are involved in translation and assembly of respiratory complexes

    Tetsuya Kotani, Shiori Akabane, Kunio Takeyasu, Takuya Ueda, Nono Takeuchi

    Nucleic Acids Research   41 ( 6 ) 3713 - 3722  2013年04月  [査読有り]

     概要を見る

    The bacterial homologues of ObgH1 and Mtg1, ObgE and RbgA, respectively, have been suggested to be involved in the assembly of large ribosomal subunits. We sought to elucidate the functions of ObgH1 and Mtg1 in ribosome biogenesis in human mitochondria. ObgH1 and Mtg1 are localized in mitochondria in association with the inner membrane, and are exposed on the matrix side. Mtg1 and ObgH1 specifically associate with the large subunit of the mitochondrial ribosome in GTP-dependent manner. The large ribosomal subunit stimulated the GTPase activity of Mtg1, whereas only the intrinsic GTPase activity was detectable with ObgH1. The knockdown of Mtg1 decreased the overall mitochondrial translation activity, and caused defects in the formation of respiratory complexes. On the other hand, the depletion of ObgH1 led to the specific activation of the translation of subunits of Complex V, and disrupted its proper formation. Our results suggested that Mtg1 and ObgH1 function with the large subunit of the mitochondrial ribosome, and are also involved in both the translation and assembly of respiratory complexes. The fine coordination of ribosome assembly, translation and respiratory complex formation in mammalian mitochondria is affirmed. © The Author(s) 2013. Published by Oxford University Press.

    DOI PubMed

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    43
    被引用数
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  • Effects of Chain Length of an Amphipathic Polypeptide Carrying the Repeated Amino Acid Sequence (LETLAKA)(n) on alpha-Helix and Fibrous Assembly Formation

    Toshiaki Takei, Kazuya Hasegawa, Katsumi Imada, Keiichi Namba, Kouhei Tsumoto, Yukino Kuriki, Masakuni Yoshino, Kazumori Yazaki, Shuichi Kojima, Tsunetomo Takei, Takuya Ueda, Kin-ichiro Miura

    BIOCHEMISTRY   52 ( 16 ) 2810 - 2820  2013年04月  [査読有り]

     概要を見る

    Polypeptide alpha 3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic alpha-helix and a long fibrous assembly. Here, we investigated the ability of alpha 3-series polypeptides (with 14-42 residues) of various chain lengths to form alpha-helices and fibrous assemblies. Polypeptide alpha 2 (14 residues), with two same-sequence repeats, did not form an alpha-helix, but polypeptide alpha 2L (15 residues; alpha 2 with one additional leucine residue on its carboxyl terminal) did form an alpha-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide alpha 2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of alpha-helix and fibril formation. In contrast, polypeptides alpha 5 (35 residues) and alpha 6 (42 residues) aggregated easily, although they formed alpha-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 angstrom and had periodicities coincident with the length of the alpha-helix in a longitudinal direction. These results indicated that the alpha-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic alpha-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.

    DOI

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    5
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  • Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Thermus thermophilus HB8

    Ami Matsumoto, Yoshihiro Shimizu, Chie Takemoto, Takuya Ueda, Toshio Uchiumi, Kosuke Ito

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications   69 ( 3 ) 332 - 335  2013年03月  [査読有り]

     概要を見る

    Peptidyl-tRNA is produced from the ribosome as a result of aborted translation. Peptidyl-tRNA hydrolase cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, to recycle tRNA for further rounds of protein synthesis. In this study, peptidyl-tRNA hydrolase from Thermus thermophilus HB8 (TthPth) was crystallized using 2-methyl-2,4-pentanediol as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.45, b = 53.92, c = 58.67Å, and diffracted X-rays to atomic resolution (beyond 1.0Å resolution). The asymmetric unit is expected to contain one TthPth molecule, with a solvent content of 27.13% (V M = 1.69Å3Da-1). The structure is being solved by molecular replacement. © 2013 International Union of Crystallography All rights reserved.

    DOI PubMed

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  • Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Thermus thermophilus HB8

    Ami Matsumoto, Yoshihiro Shimizu, Chie Takemoto, Takuya Ueda, Toshio Uchiumi, Kosuke Ito

    Acta Crystallographica Section F: Structural Biology and Crystallization Communications   69 ( 3 ) 332 - 335  2013年03月  [査読有り]

     概要を見る

    Peptidyl-tRNA is produced from the ribosome as a result of aborted translation. Peptidyl-tRNA hydrolase cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, to recycle tRNA for further rounds of protein synthesis. In this study, peptidyl-tRNA hydrolase from Thermus thermophilus HB8 (TthPth) was crystallized using 2-methyl-2,4-pentanediol as a precipitant. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 47.45, b = 53.92, c = 58.67Å, and diffracted X-rays to atomic resolution (beyond 1.0Å resolution). The asymmetric unit is expected to contain one TthPth molecule, with a solvent content of 27.13% (V M = 1.69Å3Da-1). The structure is being solved by molecular replacement. © 2013 International Union of Crystallography All rights reserved.

    DOI PubMed

    Scopus

    1
    被引用数
    (Scopus)
  • Type 1 IFN inhibits the growth factor deprived apoptosis of cultured human aortic endothelial cells and protects the cells from chemically induced oxidative cytotoxicity

    Emiko Sano, Shinnya Tashiro, Hisashi Tadakuma, Toshiaki Takei, Takuya Ueda, Kouhei Tsumoto

    JOURNAL OF CELLULAR BIOCHEMISTRY   113 ( 12 ) 3823 - 3834  2012年12月  [査読有り]

     概要を見る

    It has been shown that the genesis of atherosclerotic lesions is resulted from the injury of vascular endothelial cells and the cell damage is triggered by oxygen radicals generated from various tissues. Human vascular endothelial cells can survive and proliferate depending on growth factors such as VEGF or basic FGF and are induced apoptosis by the deprivation of growth factor or serum. It was found that type 1 IFN inhibits the growth factor deprived cell death of human aortic endothelial cells (HAEC) and protects the cells from chemically induced oxidative cytotoxicity. The anti-apoptotic effects of type 1 IFN were certified by flow cytometry using annexin-V-FITC/PI double staining and cell cycle analysis, fluorescence microscopy using Hoechst33342 and PI, colorimetric assay for caspase-3 activity, p53 and bax mRNA expressions, and cell counts. It was considered that IFN-beta inhibits the executive late stage apoptosis from the results of annexin-V-FITC/PI double staining and the inhibition of caspase-3 activity, and that the anti-apoptotic effect might be owing to the direct inhibition of the apoptotic pathway mediated by p53 from the transient down-regulation of bax mRNA expression. Whereas, type 1 IFN protected the cells from the oxidative cytotoxicity induced by tertiary butylhydroperoxide (TBH) under the presence of Ca2+. The effects of IFN-beta is more potent inhibitor of cell death than IFN-a. These results indicate that type 1 IFN, especially IFN-beta may be useful for the diseases with vascular endothelium damage such as atherosclerosis or restenosis after angioplasty as a medical treatment or a prophylactic. J. Cell. Biochem. 113: 38233834, 2012. (C) 2012 Wiley Periodicals, Inc.

    DOI

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    6
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  • Structural basis for the substrate recognition and catalysis of peptidyl-tRNA hydrolase

    Kosuke Ito, Ryo Murakami, Masahiro Mochizuki, Hao Qi, Yoshihiro Shimizu, Kin-ichiro Miura, Takuya Ueda, Toshio Uchiumi

    NUCLEIC ACIDS RESEARCH   40 ( 20 ) 10521 - 10531  2012年11月  [査読有り]

     概要を見る

    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are produced by aborted translation, to recycle tRNA for further rounds of protein synthesis. Pth is ubiquitous in nature, and its enzymatic activity is essential for bacterial viability. We have determined the crystal structure of Escherichia coli Pth in complex with the tRNA CCA-acceptor-T Psi C domain, the enzyme-binding region of the tRNA moiety of the substrate, at 2.4 A resolution. In combination with site-directed mutagenesis studies, the structure identified the amino acid residues involved in tRNA recognition. The structure also revealed that Pth interacts with the tRNA moiety through the backbone phosphates and riboses, and no base-specific interactions were observed, except for the interaction with the highly conserved base G53. This feature enables Pth to accept the diverse sequences of the elongator-tRNAs as substrate components. Furthermore, we propose an authentic Pth:peptidyl-tRNA complex model and a detailed mechanism for the hydrolysis reaction, based on the present crystal structure and the previous studies' results.

    DOI

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    25
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  • Robust in vitro affinity maturation strategy based on interface-focused high-throughput mutational scanning

    Yasuhiro Fujino, Risako Fujita, Kouichi Wada, Kotomi Fujishige, Takashi Kanamori, Lindsey Hunt, Yoshihiro Shimizu, Takuya Ueda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   428 ( 3 ) 395 - 400  2012年11月  [査読有り]

     概要を見る

    Development of protein therapeutics or biosensors often requires in vitro affinity maturation. Here we report a robust affinity engineering strategy using a custom designed library. The strategy consists of two steps beginning with identification of beneficial single amino acid substitutions then combination. A high quality combinatorial library specifically customized to a given binding-interface can be rapidly designed by high-throughput mutational scanning of single substitution libraries. When applied to the optimization of a model antibody Fab fragment, the strategy created a diverse panel of high affinity variants. The most potent variant achieved 2110-fold affinity improvement to an equilibrium dissociation constant (Kd) of 3.45 pM with only 7 amino acid substitutions. The method should facilitate affinity engineering of a wide variety of protein-protein interactions due to its context-dependent library design strategy. (C) 2012 Elsevier Inc. All rights reserved.

    DOI

    Scopus

    28
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  • Crystal structure analysis of the translation factor RF3 (release factor 3)

    Kiyohito Kihira, Yoshihiro Shimizu, Yasuhito Shomura, Naoki Shibata, Masaya Kitamura, Atsushi Nakagawa, Takuya Ueda, Kozo Ochi, Yoshiki Higuchi

    FEBS LETTERS   586 ( 20 ) 3705 - 3709  2012年10月  [査読有り]

     概要を見る

    The bacterial translational GTPases release factor RF3 promotes translation termination by recycling RF1 or RF2. Here, we present the crystal structures of RF3 complexed with GDP and guanosine 3',5'-(bis)diphosphate (ppGpp) at resolutions of 1.8 and 3.0 angstrom, respectively. ppGpp is involved in the socalled "stringent response" of bacteria. ppGpp binds at the same site as GDP, suggesting that GDP and ppGpp are two alternative physiologically relevant ligands of RF3. We also found that ppGpp decelerates the recycling of RF1 by RF3. These lines of evidence suggest that RF3 functions both as a cellular metabolic sensor and as a regulator. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI

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    24
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  • Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

    Takanori Nagano, Kouji Kojima, Toru Hisabori, Hidenori Hayashi, Eugene Hayato Morita, Takashi Kanamori, Tomoko Miyagi, Takuya Ueda, Yoshitaka Nishiyama

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 34 ) 28697 - 28704  2012年08月  [査読有り]

     概要を見る

    Elongation factor G (EF-G), a key protein in translational elongation, is known to be particularly susceptible to oxidation in Escherichia coli. However, neither the mechanism of the oxidation of EF-G nor the influence of its oxidation on translation is fully understood. In the present study, we investigated the effects of oxidants on the chemical properties and function of EF-G using a translation system in vitro derived from E. coli. Treatment of EF-G with 0.5 mM H2O2 resulted in the complete loss of translational activity. The inactivation of EF-G by H2O2 was attributable to the oxidation of two specific cysteine residues, namely, Cys(114) and Cys(266), and subsequent formation of an intramolecular disulfide bond. Replacement of Cys(114) by serine rendered EF-G insensitive to oxidation and inactivation by H2O2. Furthermore, generation of the translation system in vitro with the mutated EF-G protected the entire translation system from oxidation, suggesting that EF-G might be a primary target of oxidation within the translation system. Oxidized EF-G was reactivated via reduction of the disulfide bond by thioredoxin, a ubiquitous protein that mediates dithiol-disulfide exchange. Our observations indicate that the translational machinery in E. coli is regulated, in part, by the redox state of EF-G, which might depend on the balance between the supply of reducing power and the degree of oxidative stress.

    DOI

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    20
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  • Target Specificity of an Autoreactive Pathogenic Human gamma delta-T Cell Receptor in Myositis

    Jessica Bruder, Katherina Siewert, Birgit Obermeier, Joachim Malotka, Peter Scheinert, Josef Kellermann, Takuya Ueda, Reinhard Hohlfeld, Klaus Dornmair

    JOURNAL OF BIOLOGICAL CHEMISTRY   287 ( 25 ) 20986 - 20995  2012年06月  [査読有り]

     概要を見る

    In polymyositis and inclusion body myositis, muscle fibers are surrounded and invaded by CD8-positive cytotoxic T cells expressing the alpha beta-T cell receptor (alpha beta-TCR) for antigen. In a rare variant of myositis, muscle fibers are similarly attacked by CD8-negative T cells expressing the gamma delta-TCR (gamma delta-T cell-mediated myositis). We investigated the antigen specificity of a human gamma delta-TCR previously identified in an autoimmune tissue lesion of gamma delta-T cell-mediated myositis. We show that this V gamma 1.3V delta 2-TCR, termed M88, recognizes various proteins from different species. Several of these proteins belong to the translational apparatus, including some bacterial and human aminoacyl-tRNA synthetases (AA-RS). Specifically, M88 recognizes histidyl-tRNA synthetase, an antigen known to be also targeted by autoantibodies called anti-Jo-1. The M88 target epitope is strictly conformational, independent of post-translational modification, and exposed on the surface of the respective antigenic protein. Extensive mutagenesis of the translation initiation factor-1 from Escherichia coli (EcIF1), which served as a paradigm antigen with known structure, showed that a short alpha-helical loop around amino acids 39 to 42 of EcIF1 is a major part of the M88 epitope. Mutagenesis of M88 showed that the complementarity determining regions 3 of both gamma delta-TCR chains contribute to antigen recognition. M88 is the only known example of a molecularly characterized gamma delta-TCR expressed by autoaggressive T cells in tissue. The observation that AA-RS are targeted by a gamma delta-T cell and by autoantibodies reveals an unexpected link between T cell and antibody responses in autoimmune myositis.

    DOI

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    40
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  • Global analysis of chaperone effects using a reconstituted cell-free translation system

    Tatsuya Niwa, Takashi Kanamori, Takuya Ueda, Hideki Taguchi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   109 ( 23 ) 8937 - 8942  2012年06月  [査読有り]

     概要を見る

    Protein folding is often hampered by protein aggregation, which can be prevented by a variety of chaperones in the cell. A dataset that evaluates which chaperones are effective for aggregation-prone proteins would provide an invaluable resource not only for understanding the roles of chaperones, but also for broader applications in protein science and engineering. Therefore, we comprehensively evaluated the effects of the major Escherichia coli chaperones, trigger factor, DnaK/DnaJ/GrpE, and GroEL/GroES, on similar to 800 aggregation-prone cytosolic E. coli proteins, using a reconstituted chaperone-free translation system. Statistical analyses revealed the robustness and the intriguing properties of chaperones. The DnaK and GroEL systems drastically increased the solubilities of hundreds of proteins with weak biases, whereas trigger factor had only a marginal effect on solubility. The combined addition of the chaperones was effective for a subset of proteins that were not rescued by any single chaperone system, supporting the synergistic effect of these chaperones. The resource, which is accessible via a public database, can be used to investigate the properties of proteins of interest in terms of their solubilities and chaperone effects.

    DOI

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    132
    被引用数
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  • Traveling Time of a Translating Ribosome along Messenger RNA Monitored Directly on a Quartz Crystal Microbalance

    Shuntaro Takahashi, Kentaro Tsuji, Takuya Ueda, Yoshio Okahata

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   134 ( 15 ) 6793 - 6800  2012年04月  [査読有り]

     概要を見る

    During translation, the biosynthesis of polypeptides is dynamically regulated. The translation rate along messenger RNA (mRNA), which is dependent on the codon, structure, and sequence, is not always constant. However, methods for measuring the duration required for polypeptide elongation on an mRNA of interest have not been developed. In this work, we used a quartz crystal microbalance (QCM) technique to monitor mRNA translation in an Escherichia coli cell-free translation system in real time. This method permitted us to evaluate the translation of proteins of interest fused upstream of a streptavidin-binding peptide (SBP) fusion protein. The translation of mRNA encoding the SBP fusion protein alone was observed as a mass increase on a streptavidin-modified QCM plate. Addition of the protein of interest resulted in a delay in the mass change corresponding to the traveling time of the ribosome along the coding region of the protein of interest. With this technique, the lengths of coding sequences, codon usages, influences of unique sequences, and various protein-coding sequences were evaluated. The results showed that the traveling time of the translating ribosome depends on the length of the coding region translated but is also affected by the sequence itself. Differences in the time lags for various proteins imply that mRNA coding sequences may regulate gene expression.

    DOI

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    17
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  • Functional analysis of membranous F-0-a subunit of F1F0-ATP synthase by in vitro protein synthesis

    Yutetsu Kuruma, Toshiharu Suzuki, Sakurako Ono, Masasuke Yoshida, Takuya Ueda

    BIOCHEMICAL JOURNAL   442 ( 3 ) 631 - 638  2012年03月  [査読有り]

     概要を見る

    The a subunit of F1F0 (F1F0-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H+-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F-0 a-less F1F0 complex in Escherichia coli membrane vesicles and liposomes. The resulting F1F0 has a H+-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F1F0. By using this procedure, we analysed five mutations of F1F0, where the conserved residues in the a subunit (Asn(90), Asp(112), Arg(169), Asn(173) and Gln(217)) were individually replaced with alanine. All of the mutant F(0)a subunits were successfully incorporated into F1F0 showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in FIR,. The NI 73A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn(90) is located in the middle of putative second transmembrane helix and likely to play an important role in H+-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

    DOI

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    43
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  • Functional analysis of membranous F-0-a subunit of F1F0-ATP synthase by in vitro protein synthesis

    Yutetsu Kuruma, Toshiharu Suzuki, Sakurako Ono, Masasuke Yoshida, Takuya Ueda

    BIOCHEMICAL JOURNAL   442   631 - 638  2012年03月  [査読有り]

     概要を見る

    The a subunit of F1F0 (F1F0-ATP synthase) is a highly hydrophobic protein with five putative transmembrane helices which plays a central role in H+-translocation coupled with ATP synthesis/hydrolysis. In the present paper, we show that the a subunit produced by the in vitro protease-free protein synthesis system (the PURE system) is integrated into a preformed F-0 a-less F1F0 complex in Escherichia coli membrane vesicles and liposomes. The resulting F1F0 has a H+-coupled ATP synthesis/hydrolysis activity that is approximately half that of the native F1F0. By using this procedure, we analysed five mutations of F1F0, where the conserved residues in the a subunit (Asn(90), Asp(112), Arg(169), Asn(173) and Gln(217)) were individually replaced with alanine. All of the mutant F(0)a subunits were successfully incorporated into F1F0 showing the advantage over conventional expression in E. coli by which three (N90A, D112A, and Q217A) mutant a subunits were not found in FIR,. The NI 73A mutant retained full activity and the mutants D112A and Q217A had weak, but detectable, activity. No activity was observed for the R169A and N90A mutants. Asn(90) is located in the middle of putative second transmembrane helix and likely to play an important role in H+-translocation. The present study exemplifies that the PURE system provides an alternative approach when in vivo expression of membranous components in protein complexes turns out to be difficult.

    DOI

    Scopus

    43
    被引用数
    (Scopus)
  • 3F0912 Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer(Membrane Proteins,Oral Presentation)

    Kawai Takeaki, Caaveiro Jose, Katagiri Toyomasa, Tadakuma Hisashi, Ueda Takuya, Tsumoto Kouhei

    生物物理   52   S65 - S66  2012年

    DOI CiNii

  • 1B1510 再構築型生体外タンパク質合成系を用いた分子シャペロンによる凝集抑制効果の大規模解析(蛋白質-構造機能相関I,口頭発表,日本生物物理学会第50回年会(2012年度))

    Niwa Tatsuya, Ueda Takuya, Taguchi Hideki

    生物物理   52   S21 - S22  2012年

    DOI CiNii

  • 1PS042 多分子キネシン間の協調性は運搬物を効率的に長距離輸送するのに重要である(日本生物物理学会第50回年会(2012年度))

    Miyazono Yuya, Endo Masayuki, Ueda Takuya, Sugiyama Hiroshi, Harada Yoshie, Tadakuma Hisashi

    生物物理   52   S81  2012年

    DOI CiNii

  • 3PT111 DNAナノ構造を用いたDNA-RNAポリメラーゼ・ハイプリッドナノマシンの構築と機能評価(日本生物物理学会第50回年会(2012年度))

    Masubuchi Takeya, Tadakuma Hisashi, Endo Masayuki, Sugiyama Hiroshi, Harada Yoshie, Ueda Takuya

    生物物理   52   S159  2012年

    DOI CiNii

  • Peptide screening using pure ribosome display

    Hiroyuki Ohashi, Takashi Kanamori, Eriko Osada, Bintang K. Akbar, Takuya Ueda

    Methods in Molecular Biology   805   251 - 259  2012年  [査読有り]

     概要を見る

    To demonstrate directed protein evolution or selection of functional polypeptides, ribosome display is one of the most ideal technologies of evolutionary engineering. Intrinsic components, such as nucleases in the cell extract-based cell-free protein synthesis systems, reduce the stability of the messenger RNA-ribosome-polypeptide ternary complex, thereby preventing the attainment of reliable results. To overcome this problem, we have developed an effective and highly controllable ribosome display system using the protein synthesizing using recombinant elements (PURE) system. Since the activities of nucleases and other inhibitory factors are very low in the PURE system, the ternary complex is highly stable and the selected mRNA can be reliably recovered. Using this system, we were able to select peptides that specifically bind to monoclonal antibodies from random peptide libraries. The advantages of the modified PURE system for ribosome display strongly substantiate its usability. © 2012 Springer Science+Business Media, LLC.

    DOI PubMed

    Scopus

    13
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  • Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Escherichia coli in complex with the acceptor-T?C domain of tRNA

    Kosuke Ito, Hao Qi, Yoshihiro Shimizu, Ryo Murakami, Kin-ichiro Miura, Takuya Ueda, Toshio Uchiumi

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   67 ( Pt 12 ) 1566 - 1569  2011年12月  [査読有り]

     概要を見る

    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the product of aborted translation. In the present work, Pth from Escherichia coli was crystallized with the acceptor-TC domain of tRNA using 1,4-butanediol as a precipitant. The crystals belonged to the hexagonal space group P61, with unit-cell parameters a=b = 55.1, c = 413.1 angstrom, and diffracted X-rays beyond 2.4 angstrom resolution. The asymmetric unit is expected to contain two complexes of Pth and the acceptor-TC domain of tRNA (VM = 2.8 angstrom 3 Da-1), with a solvent content of 60.8%. The structure is being solved by molecular replacement.

    DOI

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    9
    被引用数
    (Scopus)
  • Creation of biosystems in a testtube

    Takuya Ueda

    GENES & GENETIC SYSTEMS   86 ( 6 ) 395 - 395  2011年12月  [査読有り]

  • Crystallization and preliminary X-ray analysis of peptidyl-tRNA hydrolase from Escherichia coli in complex with the acceptor-T?C domain of tRNA

    Kosuke Ito, Hao Qi, Yoshihiro Shimizu, Ryo Murakami, Kin-ichiro Miura, Takuya Ueda, Toshio Uchiumi

    ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS   67   1566 - 1569  2011年12月  [査読有り]

     概要を見る

    Peptidyl-tRNA hydrolase (Pth) cleaves the ester bond between the peptide and the tRNA of peptidyl-tRNA molecules, which are the product of aborted translation. In the present work, Pth from Escherichia coli was crystallized with the acceptor-TC domain of tRNA using 1,4-butanediol as a precipitant. The crystals belonged to the hexagonal space group P61, with unit-cell parameters a=b = 55.1, c = 413.1 angstrom, and diffracted X-rays beyond 2.4 angstrom resolution. The asymmetric unit is expected to contain two complexes of Pth and the acceptor-TC domain of tRNA (VM = 2.8 angstrom 3 Da-1), with a solvent content of 60.8%. The structure is being solved by molecular replacement.

    DOI

    Scopus

    9
    被引用数
    (Scopus)
  • Amphiphilic Polysaccharide Nanogels as Artificial Chaperones in Cell-Free Protein Synthesis

    Yoshihiro Sasaki, Wakiko Asayama, Tatsuya Niwa, Shin-ichi Sawada, Takuya Ueda, Hideki Taguchi, Kazunari Akiyoshi

    MACROMOLECULAR BIOSCIENCE   11 ( 6 ) 814 - 820  2011年06月  [査読有り]

     概要を見る

    Cell-free protein synthesis is a promising technique for the rapid production of proteins. However, the application of the cell-free systems requires the development of an artificial chaperone that prevents aggregation of the protein and supports its correct folding. Here, nanogel-based artificial chaperones are introduced that improve the folding efficiency of rhodanese produced in cell-free systems. Although rhodanese suffers from rapid aggregation, rhodanese was successfully expressed in the presence of the nanogel and folded to the enzymatically active form after addition of cyclodextrin. To validate the general applicability, the cell-free synthesis of ten water-soluble proteins was examined. It is concluded that the nanogel enables efficient expression of proteins with strong aggregation tendency.

    DOI

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    31
    被引用数
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  • Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome

    Zhan-Ping Zhou, Yoshihiro Shimizu, Hisashi Tadakuma, Hideki Taguchi, Koichi Ito, Takuya Ueda

    JOURNAL OF BIOCHEMISTRY   149 ( 5 ) 609 - 618  2011年05月  [査読有り]

     概要を見る

    Trans-translation is an eubacterial quality control system to rescue the stalled ribosome, in which multiple components such as transfer messenger RNA (tmRNA) and Small protein B (SmpB) are involved. However, how these molecules interact with ribosome remains elusive. Here, we report the single molecule analysis of the trans-translation process. We developed a new method to label the functional ribosome, in which a tag protein (the HaloTag protein of 297 amino acids) was fused to the 30S ribosomal protein S2 and covalently labelled with specific ligand (HaloTag ligand), resulting in the stable and specific labelling of ribosome. Ribosomes were anchored onto the glass surface using biotinylated derivative of the Cy3 HaloTag ligand (i.e. biotin-Cy3-ligand), and real-time interactions of Cy5-tmRNA/SmpB/EF-Tu ternary complexes with anchored ribosomes are observed as a model of the trans-translation entry. Statistical analysis revealed that Cy5-tmRNA/SmpB/EF-Tu ternary complexes bind to the anchored ribosome with the second-order rate constant of 2.6 x 10(6) (1/M/s) and tmRNAs undergo multi-modal pathway before release from ribosome. The methods presented here are also applicable to the analysis for general translation processes.

    DOI

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    20
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  • Recruitment of a species-specific translational arrest module to monitor different cellular processes

    Shinobu Chiba, Takashi Kanamori, Takuya Ueda, Yoshinori Akiyama, Kit Pogliano, Koreaki Ito

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   108 ( 15 ) 6073 - 6078  2011年04月  [査読有り]

     概要を見る

    Nascent chain-mediated translation arrest serves as a mechanism of gene regulation. A class of regulatory nascent polypeptides undergoes elongation arrest in manners controlled by the dynamic behavior of the growing chain; Escherichia coli SecM monitors the Sec protein export pathway and Bacillus subtilis MifM monitors the YidC membrane protein integration/folding pathway. We show that MifM and SecM interact with the ribosome in a species-specific manner to stall only the ribosome from the homologous species. Despite this specificity, MifM is not exclusively designed to monitor membrane protein integration because it can be converted into a secretion monitor by replacing the N-terminal transmembrane sequence with a secretion signal sequence. These results show that a regulatory nascent chain is composed of two modular elements, one devoted to elongation arrest and another devoted to subcellular targeting, and they imply that physical pulling force generated by the latter triggers release of the arrest executed by the former. The combinatorial nature may assure common occurrence of nascent chain-mediated regulation.

    DOI

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    55
    被引用数
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  • Application of micro-reactor chip technique for millisecond quenching of deuterium incorporation into 70S ribosomal protein complex

    Tatsuya Yamamoto, Yoshihiro Shimizu, Takuya Ueda, Yoshitsugu Shiro, Makoto Suematsu

    INTERNATIONAL JOURNAL OF MASS SPECTROMETRY   302 ( 1-3 ) 132 - 138  2011年04月  [査読有り]

     概要を見る

    The hydrogen/deuterium exchange (HDX) method is useful to analyze kinetics of large macromolecular complexes, although its time resolution requires further improvement. A newly developed micro-reactor chip was made of polydimethylsiloxane with a 100-mu m deep and wide microchannel. The channel in the chip has two mixing points of Y-shaped flow and allowed us to shorten time durations from the start to quenching for the HDX in 70S ribosome with high temporal resolution. This device enabled us to quench the deuterium incorporation at as early as 20 ms, detecting structural changes of individual ribosomal proteins in solution at the time scale comparable to a single reaction cycle for the peptide elongation. The profile of deuterium incorporation in individual proteins of the complex was superimposed on the X-ray crystal structure to depict the surface HDX map, revealing localization of protein movement in the ribosome. The current method serves as a useful method to visualize the regional movement of large macromolecules with high temporal resolution sufficient to examine protein dynamics. (C) 2010 Elsevier B.V. All rights reserved.

    DOI

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    8
    被引用数
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  • Protein Generation Using a Reconstituted System

    Bei-Wen Ying, Takuya Ueda

    Protein Engineering Handbook, Volume 1 &amp; Volume 2   2   515 - 535  2011年03月  [査読有り]

    DOI

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    2
    被引用数
    (Scopus)
  • Crystal structure analysis of release factor 3

    Takuya Ueda

    Acta Crystallographica Section A: Foundations and Advances    2011年

    DOI

  • 1M1524 DNA-kinesinハイブリッド・ナノマシンの構築(分子モーター2,第49回日本生物物理学会年会)

    Miyazono Yuya, Endo Masayuki, Ueda Takuya, Sugiyama Hiroshi, Harada Yoshie, Tadakuma Hisashi

    生物物理   51   S64 - S65  2011年

    DOI CiNii

  • Artificial organelle for energy production in artificial cell.

    Yutetsu Kuruma, Toshiharu Suzuki, Masasuke Yoshida, Takuya Ueda

    Advances in Artificial Life: 20th Anniversary Edition - Back to the Origins of Alife, ECAL 2011, Paris, France, August 8-12, 2011     438  2011年  [査読有り]

  • The finite element method analysis for the stress intensity factors using a path independent Ê-integral formula

    Yatomi C, Ueda T, Takagi S, Abe T

    Zairyo/Journal of the Society of Materials Science, Japan   60 ( 11 ) 1031 - 1036  2011年  [査読有り]

    DOI

    Scopus

  • Analysis of the functional consequences of lethal mutations in mitochondrial translational elongation factors

    Kenta Akama, Brooke E. Christian, Christie N. Jones, Takuya Ueda, Nono Takeuchi, Linda L. Spremulli

    BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE   1802 ( 7-8 ) 692 - 698  2010年07月  [査読有り]

     概要を見る

    Mammalian mitochondria synthesize a set of thirteen proteins that are essential for energy generation via oxidative phosphorylation. The genes for all of the factors required for synthesis of the mitochondrially encoded proteins are located in the nuclear genome. A number of disease-causing mutations have been identified in these genes. In this manuscript, we have elucidated the mechanisms of translational failure for two disease states characterized by lethal mutations in mitochondrial elongation factor Ts (EF-Ts-mt) and elongation factor Tu (EF-Tu(mt)). EF-Tu(mt) delivers the aminoacyl-tRNA (aa-tRNA) to the ribosome during the elongation phase of protein synthesis. EF-Ts-mt regenerates EF-Tu(mt):GTP from EF-Tu(mt):GDP. A mutation of EF-Ts-mt (R325W) leads to a two-fold reduction in its ability to stimulate the activity of EF-Tu(mt) in poly(U)directed polypeptide chain elongation. This loss of activity is caused by a significant reduction in the ability of EF-Ts-mt R325W to bind EF-Tu(mt), leading to a defect in nucleotide exchange. A mutation of Arg336 to Gin in EF-Tu(mt) causes infantile encephalopathy caused by defects in mitochondrial translation. EF-Tu(mt) R336Q is as active as the wild-type protein in polymerization using Escherichia coli 70S ribosomes and E. coli [C-14]Phe-tRNA but is inactive in polymerization with mitochondrial [C-14]Phe-tRNA and mitochondrial 555 ribosomes. The R336Q mutation causes a two-fold decrease in ternary complex formation with E coli aa-tRNA but completely inactivates EF-Tu(mt) for binding to mitochondrial aa-tRNA. Clearly the R336Q mutation in EF-Tu(mt) has a far more drastic effect on its interaction with mitochondrial aa-tRNAs than bacterial aa-tRNAs. (C) 2010 Elsevier B.V. All rights reserved.

    DOI

    Scopus

    11
    被引用数
    (Scopus)
  • A novel complete reconstitution system for membrane integration of the simplest membrane protein

    Ken-ichi Nishiyama, Masahide Maeda, Masato Abe, Takashi Kanamori, Keiko Shimamoto, Shoichi Kusumoto, Takuya Ueda, Hajime Tokuda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   394 ( 3 ) 733 - 736  2010年04月  [査読有り]

     概要を見る

    A complete reconstitution system for membrane integration of the simplest protein was developed by means of defined factors A mutant version of Pf3 coat protein, 3L-Pf3 coat, requires neither signal recognition particle/Sec factors nor a membrane potential for its integration into the cytoplasmic membrane of Escherichia colt Although 3L-Pf3 coat is spontaneously integrated into liposomes composed of phospholipids, diacylglycerol completely blocks such spontaneous integrations at a physiological level Under the conditions where spontaneous integration does not occur, 3L-Pf3 coat integration was absolutely dependent on a novel integration-stimulating factor. Combination of the PURE system, an in vitro translation system composed of the purified factors involved in translation in E. coli, with liposomes containing the highly purified integration-stimulating factor revealed multiple cycles of 3L-Pf3 coat integration, achieving the complete reconstitution of membrane integration Based on the function of the factor, we propose that the factor is named MPlase (Membrane Protein Integrase). (C) 2010 Elsevier Inc All rights reserved

    DOI

    Scopus

    44
    被引用数
    (Scopus)
  • A Highly Controllable Reconstituted Cell-Free System -a Breakthrough in Protein Synthesis Research

    Hiroyuki Ohashi, Takashi Kanamori, Yoshihiro Shimizu, Takuya Ueda

    CURRENT PHARMACEUTICAL BIOTECHNOLOGY   11 ( 3 ) 267 - 271  2010年04月  [査読有り]

     概要を見る

    The PURE system is a highly controllable cell-free protein synthesis system composed of individually prepared components that are required for protein synthesis in Escherichia coli. The PURE system contains neither nucleases nor proteases, both of which degrade DNA or mRNA templates and proteins. The protein products are easily purified using affinity chromatography to remove the tagged protein factors. The PURE system should help to create new fields in protein research.

    DOI

    Scopus

    50
    被引用数
    (Scopus)
  • A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

    Takuma Suematsu, Shin-ichi Yokobori, Hiroyuki Morita, Shigeo Yoshinari, Takuya Ueda, Kiyoshi Kita, Nono Takeuchi, Yoh-ichi Watanabe

    MOLECULAR MICROBIOLOGY   75 ( 6 ) 1445 - 1454  2010年03月  [査読有り]

     概要を見る

    P&gt;Translation elongation factor G (EF-G) in bacteria plays two distinct roles in different phases of the translation system. EF-G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post-termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF-G are distributed over two different EF-G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF-G is also found in bacteria. Two EF-G paralogues are found in the spirochaete Borrelia burgdorferi, EF-G1 and EF-G2. We demonstrate that EF-G1 is a translocase, while EF-G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF-G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF-3) is present in the reaction. These results indicate that two B. burgdorferi EF-G paralogues are close relatives to mitochondrial EF-G paralogues rather than the conventional bacterial EF-G, in both their phylogenetic and biochemical features.

    DOI

    Scopus

    22
    被引用数
    (Scopus)
  • Mg2+ Dependence of 70 S Ribosomal Protein Flexibility Revealed by Hydrogen/Deuterium Exchange and Mass Spectrometry

    Tatsuya Yamamoto, Yoshihiro Shimizu, Takuya Ueda, Yoshitsugu Shiro

    JOURNAL OF BIOLOGICAL CHEMISTRY   285 ( 8 ) 5646 - 5652  2010年02月  [査読有り]

     概要を見る

    The ribosome from Escherichia coli requires a specific concentration of Mg2+ to maintain the 70 S complex formation and allow protein synthesis, and then the structure must be stable and flexible. How does the ribosome acquire these conflicting factors at the same time? Here, we investigated the hydrogen/deuterium exchange of 52 proteins in the 70 S ribosome, which controlled stability and flexibility under various Mg2+ concentrations, using mass spectrometry. Many proteins exhibited a sigmoidal curve for Mg2+ concentration dependence, incorporating more deuterium at lower Mg2+ concentration. By comparing deuterium incorporation with assembly, we have discovered a typical mechanism of complexes for acquiring both stability and flexibility at the same time. In addition, we got information of the localization of flexibility in ribosomal function by the analysis of related proteins with stalk protein, tRNA, mRNA, and nascent peptide, and demonstrate the relationship between structure, assembly, flexibility, and function of the ribosome.

    DOI

    Scopus

    17
    被引用数
    (Scopus)
  • 1P173 1分子イメージングによるトランス-トランスレーションにおけるSmpBのC末端の機能解析(分子モーター,第48回日本生物物理学会年会)

    Zhou ZhanPing, Suto Yuta, Shimizu Yoshihiro, Tadakuma Hisashi, Taguchi Hideki, Ito Koichi, Ueda Takuya

    生物物理   50 ( 2 ) S49 - S50  2010年

    DOI CiNii

  • 1P247 1I1520 セルフリー蛋白質合成による機能性生体膜の構築(生体膜・人工膜-ダイナミクス,口頭発表,第48回日本生物物理学会年会)

    Yutetsu Kuruma, Suzuki Toshiharu, Yoshida Masasuke, Ueda Takuya

    生物物理   50 ( 2 ) S63  2010年

    DOI CiNii

  • 2P128 転写・翻訳系がカップルしたin vitro無細胞翻訳系再構成の試み(核酸結合蛋白質,第48回日本生物物理学会年会)

    Tanaka Koji, Matsui Jun, Koyama Hiroshi, Tamaru Daichi, Tadakuma Hisashi, Shimizu Yoshihiro, Ueda Takuya

    生物物理   50 ( 2 ) S104 - S105  2010年

    DOI CiNii

  • Ribosome Display with the PURE Technology

    Ueda Takuya, Kanamori Takashi, Ohashi Hiroyuki

    Cell-Free Protein Production: Methods and Protocols   607   219 - 225  2010年  [査読有り]  [国際誌]

    DOI PubMed

    Scopus

    21
    被引用数
    (Scopus)
  • Production of multi-subunit complexes on liposome through an E. coli cell-free expression system.

    Kuruma, Y., Suzuki, T., Ueda, T.

    Methods in molecular biology (Clifton, N.J.)   607   161 - 171  2010年  [査読有り]

    DOI

    Scopus

    17
    被引用数
    (Scopus)
  • PURE technology.

    Shimizu, Y., Ueda, T.

    Methods in molecular biology (Clifton, N.J.)   607   11 - 21  2010年  [査読有り]

    DOI

    Scopus

    88
    被引用数
    (Scopus)
  • Loss of mutant mitochondrial DNA harboring the MELAS A3243G mutation in human cybrid cells after cell-cell fusion with normal tissue-derived fibroblast cells

    Takamitsu Yano, Masashi Tanaka, Noboru Fukuda, Takuya Ueda, Hiroki Nagase

    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE   25 ( 1 ) 153 - 158  2010年01月  [査読有り]

     概要を見る

    Mutant mitochondrial (mt) DNA variants are related to human disease and have been investigated using cytoplasmic hybrid (cybrid) cells generated from human tumor cells in which mutant nit maintenance depends on the cell line. It is, however, unclear whether human intercellular fusion of non-tumorous cells influences the maintenance of disease-related mutant mt. A preliminary experiment of cell-cell fusion between a human skin fibroblast cell line from a Lesch-Nyhan syndrome patient and an osteosarcoma cybrid cell line harboring the mitochondrial tRNA(Leu(UUR))A3243G mutation showed a decrease of A3243G mutant mtDNA in fused cells during passages. In order to confirm the decrease of mutant mtDNA, we performed cell-cell fusion experiments using another human lung fibroblastic cell line. When the hygromycin-resistant osteosarcoma cybrid cell line was fused with the fibroblasts without any A3243G mtDNA mutations, the proportion of A3243G mutant mtDNA in the hybrid cells gradually decreased during cell culture and almost completely disappeared in all hybrid clones at the end of 15 passages. These results indicated that A3243G mutant specific mtDNA decreases in the hybrid background when normal fibroblast-derived cell contents, including the nucleus and mt, were introduced. Thus, we are hypothesizing that the non-tumorigenic fibroblast cellular components induce a difference in replication efficacy between the mtDNAs with and without the A3243G mutant sequence, which may be related to the decrease of disease-related mutant mtDNA in the hybrid cells.

    DOI

    Scopus

    2
    被引用数
    (Scopus)
  • EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein syntheis

    Nono Takeuchi, Takuya Ueda

    Seikagaku   82 ( 9 ) 825 - 831  2010年  [査読有り]

    PubMed

  • On the origin of specific macromolecular sequences, catalytic cycles and the rna world: The 'ligand-imprinting hypothesis'

    Ueda, T.

    Origins of Life and Evolution of Biospheres   40 ( 4 ) 411 - 413  2010年  [査読有り]

  • EF-G2mt is an exclusive recycling factor in mammalian mitochondrial protein syntheis

    Nono Takeuchi, Takuya Ueda

    Seikagaku   82 ( 9 ) 825 - 831  2010年  [査読有り]

    PubMed

  • EF-G2mt Is an Exclusive Recycling Factor in Mammalian Mitochondrial Protein Synthesis

    Masafumi Tsuboi, Hiroyuki Morita, Yusuke Nozaki, Kenta Akama, Takuya Ueda, Koichi Ito, Knud H. Nierhaus, Nono Takeuchi

    MOLECULAR CELL   35 ( 4 ) 502 - 510  2009年08月  [査読有り]

     概要を見る

    Bacterial translation elongation factor G (EF-G) catalyzes translocation during peptide elongation and mediates ribosomal disassembly during ribosome recycling in concert with the ribosomal recycling factor (RRF). Two homologs of EF-G have been identified in mitochondria from yeast to man, EF-G1mt and EF-G2mt. Here, we demonstrate that the dual function of bacterial EF-G is divided between EF-G1mt and EF-G2mt in human mitochondria (RRFmt). EF-G1mt specifically catalyzes translocation, whereas EF-G2mt mediates ribosome recycling with human mitochondrial RRF but lacks translocation activity. Domain swapping experiments suggest that the functional specificity for EF-G2mt resides in domains III and IV. Furthermore, GTP hydrolysis by EF-G2mt is not necessary for ribosomal splitting, in contrast to the bacterial-recycling mode. Because EF-G2mt represents a class of translational GTPase that is involved in ribosome recycling, we propose to rename this factor mitochondrial ribosome recycling factor 2 (RRF2mt).

    DOI

    Scopus

    89
    被引用数
    (Scopus)
  • Real-Time Monitoring of Cell-Free Translation on a Quartz-Crystal Microbalance

    Shuntaro Takahashi, Masaaki Iida, Hiroyuki Furusawa, Yoshihiro Shimizu, Takuya Ueda, Yoshio Okahata

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   131 ( 26 ) 9326 - 9332  2009年07月  [査読有り]

     概要を見る

    The efficiency of protein synthesis is often regulated post-transcriptionally by sequences within the mRNA. To investigate the reactions of protein translation, we established a system that allowed real-time monitoring of protein synthesis using a cell-free translation mixture and a 27 MHz quartz-crystal microbalance (QCM). Using an mRNA that encoded a fusion polypeptide comprising the streptavidin-binding peptide (SBP) tag, a portion of Protein D as a spacer, and the SecM arrest sequence, we could follow the binding of the SBP tag, while it was displayed on the 70S ribosome, to a streptavidin-modified QCM over time. Thus, we could follow a single turnover of protein synthesis as a change in mass. This approach allowed us to evaluate the effects of different antibiotics and mRNA sequences on the different steps of translation. From the results of this study, we have determined that both the formation of the initiation complex from the 70S ribosome, mRNA, and fMet-tRNA(fmet) and the accommodation of the second aminoacyl-tRNA to the initiation complex are rate-limiting steps in protein synthesis.

    DOI

    Scopus

    22
    被引用数
    (Scopus)
  • Twin-Arginine-Dependent Translocation of SufI in the Absence of Cytosolic Helper Proteins

    Eva Holzapfel, Michael Moser, Emile Schiltz, Takuya Ueda, Jean-Michel Betton, Matthias Muller

    BIOCHEMISTRY   48 ( 23 ) 5096 - 5105  2009年06月  [査読有り]

     概要を見る

    The twin-arginine translocation (Tat) machinery present in bacterial and thylakoidal membranes is able to transport fully folded proteins. Folding of some Tat precursor proteins requires dedicated chaperones that also sequester the signal sequence during the maturation process. Whether or not signal sequence-binding chaperones are a general prerequisite for all Tat substrate proteins is not known. Here, we have studied the propensity of Tat signal sequences of Escherichia coli to interact with general chaperones and peptidyl-prolyl-cis, trans-isomerases. Site-specific photocross-linking revealed clear specificity for FK506-binding proteins. Nevertheless transport of the Tat substrate SufI into inverted inner membrane vesicles of E. coli was found to occur in the bona fide, absence of any cytosolic chaperone. Our results suggest that in E. coli, cytosolic chaperones are not essential for the twin-arginine-dependent export of cofactor-less substrates.

    DOI

    Scopus

    14
    被引用数
    (Scopus)
  • Radiofrequency Ablation of the Liver: Determination of Ablative Margin at MR Imaging with Impaired Clearance of Ferucarbotran-Feasibility Study

    Kensaku Mori, Kuniaki Fukuda, Hitoshi Asaoka, Takuya Ueda, Akira Kunimatsu, Yoshikazu Okamoto, Katsuhiro Nasu, Kiyoshi Fukunaga, Yukio Morishita, Manabu Minami

    RADIOLOGY   251 ( 2 ) 557 - 565  2009年05月  [査読有り]

     概要を見る

    Institutional review board approval and informed consent were obtained. The feasibility of magnetic resonance (MR) imaging with impaired clearance of ferucarbotran to visualize ablated liver parenchyma surrounding a tumor (ablative margin [AM]) was evaluated after radiofrequency (RF) ablation of the liver. Twenty-one patients with hepatocellular carcinomas underwent RF ablation 2-7 hours after ferucarbotran-enhanced MR imaging. On unenhanced T2*-weighted images acquired after 3-5 days, AMs appeared as hypointense rims. The AM status was related to incidence of residual or recurrent tumors. This technique is feasible for visualization of AM and prediction of residual or recurrent tumors after RF ablation of the liver.

    DOI

  • Epitope Mapping Using Ribosome Display in a Reconstituted Cell-Free Protein Synthesis System

    Eriko Osada, Yoshihiro Shimizu, Bintang K. Akbar, Takashi Kanamori, Takuya Ueda

    JOURNAL OF BIOCHEMISTRY   145 ( 5 ) 693 - 700  2009年05月  [査読有り]

     概要を見る

    Ribosome display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the ribosome display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti--Catenin (anti--Cat) mAbs, selected peptides had a homology for the partial peptide sequences of -Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and -Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs.

    DOI

    Scopus

    19
    被引用数
    (Scopus)
  • Unconventional decoding of the AUA codon as methionine by mitochondrial tRNA(Met) with the anticodon f(5)CAU as revealed with a mitochondrial in vitro translation system

    Chie Takemoto, Linda L. Spremulli, Lisa A. Benkowski, Takuya Ueda, Takashi Yokogawa, Kimitsuna Watanabe

    NUCLEIC ACIDS RESEARCH   37 ( 5 ) 1616 - 1627  2009年04月  [査読有り]

     概要を見る

    Mitochondrial (mt) tRNA(Met) has the unusual modified nucleotide 5-formylcytidine (f(5)C) in the first position of the anticodon. This tRNA must translate both AUG and AUA as methionine. By constructing an in vitro translation system from bovine liver mitochondria, we examined the decoding properties of the native mt tRNA(Met) carrying f(5)C in the anticodon compared to a transcript that lacks the modification. The native mt Met-tRNA could recognize both AUA and AUG codons as Met, but the corresponding synthetic tRNA(Met) lacking f(5)C (anticodon CAU), recognized only the AUG codon in both the codon-dependent ribosomal binding and in vitro translation assays. Furthermore, the Escherichia coli elongator tRNA(m)(Met) with the anticodon ac(4)CAU (ac(4)C 4-acetylcytidine) and the bovine cytoplasmic initiator tRNA(Met) (anticodon CAU) translated only the AUG codon for Met on mt ribosome. The codon recognition patterns of these tRNAs were the same on E. coli ribosomes. These results demonstrate that the f(5)C modification in mt tRNA(Met) plays a crucial role in decoding the nonuniversal AUA codon as Met, and that the genetic code variation is compensated by a change in the tRNA anticodon, not by a change in the ribosome. Base pairing models of f(5)C-G and f(5)C-A based on the chemical properties of f(5)C are presented.

    DOI

    Scopus

    85
    被引用数
    (Scopus)
  • Bimodal protein solubility distribution revealed by an aggregation analysis of the entire ensemble of Escherichia coli proteins

    Tatsuya Niwa, Bei-Wen Ying, Katsuyo Saito, WenZhen Jin, Shoji Takada, Takuya Ueda, Hideki Taguchi

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   106 ( 11 ) 4201 - 4206  2009年03月  [査読有り]

     概要を見る

    Protein folding often competes with intermolecular aggregation, which in most cases irreversibly impairs protein function, as exemplified by the formation of inclusion bodies. Although it has been empirically determined that some proteins tend to aggregate, the relationship between the protein aggregation propensities and the primary sequences remains poorly understood. Here, we individually synthesized the entire ensemble of Escherichia coli proteins by using an in vitro reconstituted translation system and analyzed the aggregation propensities. Because the reconstituted translation system is chaperone-free, we could evaluate the inherent aggregation propensities of thousands of proteins in a translation-coupled manner. A histogram of the solubilities, based on data from 3,173 translated proteins, revealed a clear bimodal distribution, indicating that the aggregation propensities are not evenly distributed across a continuum. Instead, the proteins can be categorized into 2 groups, soluble and aggregation-prone proteins. The aggregation propensity is most prominently correlated with the structural classification of proteins, implying that the prediction of aggregation propensity requires structural information about the protein.

    DOI

    Scopus

    226
    被引用数
    (Scopus)
  • A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

    Yutetsu Kuruma, Pasquale Stano, Takuya Ueda, Pier Luigi Luisi

    BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES   1788 ( 2 ) 567 - 574  2009年02月  [査読有り]

     概要を見る

    Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity - inside liposomes - of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within - which corresponds to the autopoietic growth mechanism. (C) 2008 Elsevier B.V. All rights reserved.

    DOI

    Scopus

    201
    被引用数
    (Scopus)
  • [Development of technologies for site-specific incorporation of non-natural amino acids into proteins and cell-free protein synthesis system. Overview].

    Yokoyama S, Endo Y, Ueda T, Tanokura M

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   54 ( 12 Suppl ) 1441 - 1442  2009年  [査読有り]

    PubMed

  • [ PURE system: a reconstituted cell-free protein synthesis system].

    Ueda, T.

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   54 ( 12 Suppl ) 1448 - 1451  2009年  [査読有り]

  • Comprehensive aggregation analysis of whole E. coli proteins for understanding of protein aggregation

    Niwa T, Ueda T, Taguchi H

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   54 ( 14 ) 1870 - 1875  2009年  [査読有り]

    CiNii

  • Low conservation and species-specific evolution of alternative splicing in humans and mice: comparative genomics analysis using well-annotated full-length cDNAs

    Jun-ichi Takeda, Yutaka Suzuki, Ryuichi Sakate, Yoshiharu Sato, Masahide Seki, Takuma Irie, Nono Takeuchi, Takuya Ueda, Mitsuteru Nakao, Sumio Sugano, Takashi Gojobori, Tadashi Imanishi

    NUCLEIC ACIDS RESEARCH   36 ( 20 ) 6386 - 6395  2008年11月  [査読有り]

     概要を見る

    Using full-length cDNA sequences, we compared alternative splicing (AS) in humans and mice. The alignment of the human and mouse genomes showed that 86% of 199 426 total exons in human AS variants were conserved in the mouse genome. Of the 20 392 total human AS variants, however, 59% consisted of all conserved exons. Comparing AS patterns between human and mouse transcripts revealed that only 431 transcripts from 189 loci were perfectly conserved AS variants. To exclude the possibility that the full-length human cDNAs used in the present study, especially those with retained introns, were cloning artefacts or prematurely spliced transcripts, we experimentally validated 34 such cases. Our results indicate that even retained-intron type transcripts are typically expressed in a highly controlled manner and interact with translating ribosomes. We found non-conserved AS exons to be predominantly outside the coding sequences (CDSs). This suggests that non-conserved exons in the CDSs of transcripts cause functional constraint. These findings should enhance our understanding of the relationship between AS and species specificity of human genes.

    DOI

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    24
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  • Characterization of the catalytic activity of the gamma-phage lysin, PlyG, specific for Bacillus anthracis

    Hitomi S. Kikkawa, Takuya Ueda, Shin-ichi Suzuki, Jiro Yasuda

    FEMS MICROBIOLOGY LETTERS   286 ( 2 ) 236 - 240  2008年09月  [査読有り]

     概要を見る

    Bacillus anthracis causes anthrax, a lethal disease affecting humans that has attracted attention due to its bioterrorism potential. PlyG is a lysin of gamma-phage, which specifically infects B. anthracis and lyses its cell wall. PlyG contains a T7 lysozyme-like amidase domain, which appears to be the catalytic domain, in the N-terminal region and has a high degree of sequence similarity with PlyL, which is an N-acetylmuramoyl-L-alanine amidase encoded by the B. anthracis genome. Here, we demonstrated that two amino acid residues of PlyG, H29 and E90, are necessary for its catalytic activity in B. anthracis. These residues are structurally analogous to residues whose mutation in T7 lysozyme abolished its catalytic activity. A C-terminal deletion mutant of PlyG lacking the core sequence for binding to B. anthracis showed completely abolished binding activity, unlike PlyL, despite high sequence similarity with PlyL in the N-terminal region. This suggests that the C-terminal binding domain, as well as the N-terminal catalytic domain, is essential for the catalytic activity of PlyG. Our observations provide new insights into the mechanism of specific catalysis of PlyG in B. anthracis and may contribute to the establishment of new methods for anthrax therapy.

    DOI

    Scopus

    29
    被引用数
    (Scopus)
  • The human mitochondrial translation release factor HMRF1L is methylated in the GGQ motif by the methyltransferase HMPrmC

    Toshihiro Ishizawa, Yusuke Nozaki, Takuya Ueda, Nono Takeuchi

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   373 ( 1 ) 99 - 103  2008年08月  [査読有り]

     概要を見る

    We have recently identified the human mitochondrial release factor, HMRF1L, which is responsible for decoding of UAA/UAG termination codons. Here, we identified human mitochondrial methyltransferase, HMPrmC, which methylates the glutamine residue in the GGQ tripeptide motif of HMRF1L. We demonstrate that HMPrmC is targeted to mitochondria and the glutamine residue in the GGQ motif of HMRF1L is methylated in vivo. HMPrmC depletion in HeLa cells leads to decreased mitochondrial translation activity in the presence of the translation fidelity antibiotic streptomycin in galactose containing medium. These results suggest that the methylation of HMRF1L by HMPrmC in human mitochondria is involved in the control of the translation termination process, probably by preventing the undesired suppression of termination codons and/or abortive termination events at sense codons under such conditions, as observed in prokaryotes and eukaryotes systems. (C) 2008 Elsevier Inc. All rights reserved.

    DOI

    Scopus

    20
    被引用数
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  • The Constructive Approach for Cell-Free Translation

    Takuya Ueda

    Cell-Free Protein Synthesis: Methods and Protocols     35 - 50  2008年07月  [査読有り]

    DOI

    Scopus

    2
    被引用数
    (Scopus)
  • Single-molecule imaging of full protein synthesis by immobilized ribosomes

    Sotaro Uemura, Ryo Iizuka, Taro Ueno, Yoshihiro Shimizu, Hideki Taguchi, Takuya Ueda, Joseph D. Puglisi, Takashi Funatsu

    NUCLEIC ACIDS RESEARCH   36 ( 12 )  2008年07月  [査読有り]

     概要を見る

    How folding of proteins is coupled to their synthesis remains poorly understood. Here, we apply single-molecule fluorescence imaging to full protein synthesis in vitro . Ribosomes were specifically immobilized onto glass surfaces and synthesis of green fluorescent protein (GFP) was achieved using modified commercial Protein Synthesis using Recombinant Elements that lacked ribosomes but contained purified factors and enzyme that are required for translation in Escherichia coli . Translation was monitored using a GFP mutant (F64L/S65T/F99S/M153T/V163A) that has a high fluorophore maturation rate and that contained the Secretion Monitor arrest sequence to prevent dissociation from the ribosome. Immobilized ribosomal subunits were labeled with Cy3 and GFP synthesis was measured by colocalization of GFP fluorescence with the ribosome position. The rate of appearance of colocalized ribosome GFP was equivalent to the rates of fluorescence appearance coupled with translation measured in bulk, and the ribosomepolypeptide complexes were stable for hours. The methods presented here are applicable to single-molecule investigation of translational initiation, elongation and cotranslational folding.

    DOI

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    35
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    (Scopus)
  • Comprehensive detection of human terminal oligo-pyrimidine (TOP) genes and analysis of their characteristics

    Riu Yamashita, Yutaka Suzuki, Nono Takeuchi, Hiroyuki Wakaguri, Takuya Ueda, Sumio Sugano, Kenta Nakai

    NUCLEIC ACIDS RESEARCH   36 ( 11 ) 3707 - 3715  2008年06月  [査読有り]

     概要を見る

    Although the knowledge accumulated on the transcriptional regulations of eukaryotes is significant, the knowledge on their translational regulations remains limited. Thus, we performed a comprehensive detection of terminal oligo-pyrimidine (TOP), which is one of the well-characterized cis-regulatory motifs for translational controls located immediately downstream of the transcriptional start sites of mRNAs. Utilizing our precise 5-end information of the full-length cDNAs, we could screen 1645 candidate TOP genes by position specific matrix search. Among them, not only 75 out of 78 ribosomal protein genes but also eight previously identified non-ribosomal-protein TOP genes were included. We further experimentally validated the translational activities of 83 TOP candidate genes. Clear translational regulations exerted on the stimulation of 12-O-tetradecanoyl-1-phorbol-13-acetate for at least 41 of them was observed, indicating that there should be a few hundreds of human genes which are subjected to regulation at translation levels via TOPs. Our result suggests that TOP genes code not only formerly characterized ribosomal proteins and translation-related proteins but also a wider variety of proteins, such as lysosome-related proteins and metabolism-related proteins, playing pivotal roles in gene expression controls in the majority of cellular mRNAs.

    DOI

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    79
    被引用数
    (Scopus)
  • HMRF1L is a human mitochondrial translation release factor involved in the decoding of the termination codons UAA and UAG

    Yusuke Nozaki, Noriko Matsunaga, Toshihiro Ishizawa, Takuya Ueda, Nono Takeuchi

    GENES TO CELLS   13 ( 5 ) 429 - 438  2008年05月  [査読有り]

     概要を見る

    While all essential mammalian mitochondrial factors involved in the initiation and elongation phases of translation have been cloned and well characterized, little is known about the factors involved in the termination process. In the present work, we report the functional analysis of human mitochondrial translation release factors (RF). Here, we show that HMRF1, which had been previously denoted as a human mitochondrial RF, was inactive in in vitro translation system, although it is a mitochondrial protein. Instead, we identified another human mitochondrial RF candidate, HMRF1L, and demonstrated that HMRF1L is indeed a mitochondrial protein that functions specifically as an RF for the decoding of mitochondrial UAA and UAG termination codons in vitro. The identification of the functional mitochondrial RF brings us much closer to a detailed understanding of the translational termination process in mammalian mitochondria as well as to the unraveling of the molecular mechanism of diseases caused by the dys-regulation of translational termination in human mitochondria.

    DOI

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    37
    被引用数
    (Scopus)
  • Polyadenylation in mammalian mitochondria: Insights from recent studies

    Takashi Nagaike, Tsutomu Suzuki, Takuya Ueda

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS   1779 ( 4 ) 266 - 269  2008年04月  [査読有り]

     概要を見る

    Polyadenylation in animal mitochondria is very unique. Unlike other systems, polyadenylation is needed to generate UAA stop codons that are not encoded in mitochondrial (mt) DNA. In some cases, polyadenylation is required for the mt tRNA maturation by editing of its 3' termini. Furthermore, recent studies on human mt poly(A) polymerase (PAP) and PNPase provide new insights and questions for the regulatory mechanism and functional role of polyadenylation in human mitochondria. (c) 2008 Elsevier B.V. All rights reserved.

    DOI

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    34
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    (Scopus)
  • 70S ribosomes bind to Shine-Dalgarno sequences without required dissociations

    Shuntaro Takahashi, Ryoko Akita, Hisao Matsuno, Hiroyuki Furusawa, Yoshihiro Shimizu, Takuya Ueda, Yoshio Okahata

    CHEMBIOCHEM   9 ( 6 ) 870 - 873  2008年04月  [査読有り]

    DOI

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    12
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  • UncI protein can mediate ring-assembly of c-subunits of FoF1-ATP synthase in vitro

    Yoko Ozaki, Toshiharu Suzuki, Yutetsu Kuruma, Takuya Ueda, Masasuke Yoshida

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   367 ( 3 ) 663 - 666  2008年03月  [査読有り]

     概要を見る

    In FoF1-ATP synthase, multimeric c-subunits are assembled to a ring (c-ring) in the membranes that rotates as protons flow across F-o. We recently reported that assembly of c-ring of Propionigenium modestum in the membranes of Escherichia coli cells required P. modestum UncI, a product of the conserved uncI gene in the FoF1 operon. However, cooperation with endogenous factors in E. coli remained unclear. Here, P. modestum c-subunit was synthesized in vitro in the presence of liposomes. When c-subunit alone was synthesized, it did not form c-ring. However, when c-subunit and P. modestum UncI were synthesized together, c-ring was formed. Fusion of the two kinds of liposomes, one containing only unassembled c-subunit and the other only UncI, resulted in gradual formation of c-ring. Thus, UncI alone can mediate in vitro post-translational c-ring assembly. (C) 2008 Elsevier Inc. All rights reserved.

    DOI

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    35
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  • 2P-126 ナノ開口基板上でのタンパク質翻訳中における高濃度tRNAの1分子ダイナミクス(核酸・相互作用,複合体,第46回日本生物物理学会年会)

    Uemura Sotaro, Shimizu Yoshihiro, Iizuka Ryo, Ueda Takuya, Akahori Rena, Shimamoto Naonobu, Tanii Takashi, Ohdomari Iwao, Puglisi Joseph, Funatsu Takashi

    生物物理   48   S94 - S95  2008年

    DOI CiNii

  • 3P-079 無細胞タンパク質合成系におけるナノゲルの分子シャペロン機能(蛋白質工学,進化工学,第46回日本生物物理学会年会)

    Asayama Wakiko, Niwa Tatuya, Ueda Takuya, Taguchi Hideki, Akiyoshi Kazunari

    生物物理   48   S139  2008年

    DOI CiNii

  • 2P-239 リン脂質の生合成過程に関わる膜蛋白質のリポソーム内合成(生体膜/人工膜・ダイナミクス,第46回日本生物物理学会年会)

    Kuruma Yutetsu, Stano Pasquale, Ueda Takuya, Luisi Pier Luigi

    生物物理   48   S112  2008年

    DOI CiNii

  • Elongation factor Tu mutants expand amino acid tolerance of protein biosynthesis system

    Yoshio Doi, Takashi Ohtsuki, Yoshihiro Shimizu, Takuya Ueda, Masahiko Sisido

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   129 ( 46 ) 14458 - 14462  2007年11月  [査読有り]

     概要を見る

    Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.

    DOI

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    88
    被引用数
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  • Ribosomal protein S1 is not essential for the trans-translation machinery

    Hao Qi, Yoshihiro Shimizu, Takuya Ueda

    JOURNAL OF MOLECULAR BIOLOGY   368 ( 3 ) 845 - 852  2007年05月  [査読有り]

     概要を見る

    In eubacteria, ribosome stalling during protein synthesis is rescued by a tmRNA-derived trans-translation system. Because ribosomal protein S1 specifically binds to tmRNA with high affinity, it is considered to be involved in the trans-translation system. However, the role of S1 in trans-translation is still unclear. To study the function of S1 in the trans-translation system, we constructed an S1-free cell-free translation system. We found that trans-translation proceeded even in the absence of S1. Addition of S1 into the S1-free system did not affect trans-translation efficiency. These results suggest that S1 does not play a role in the trans-translation machinery. (c) 2007 Elsevier Ltd. All rights reserved.

    DOI

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    25
    被引用数
    (Scopus)
  • Nuclear respiratory factor 2 activates transcription of human mitochondrial translation initiation factor 2 gene

    Rippei Hayashi, Takuya Ueda, Mary A. Farwell, Nono Takeuchi

    MITOCHONDRION   7 ( 3 ) 195 - 203  2007年05月  [査読有り]

     概要を見る

    We studied the transcriptional regulation of the human mitochondrial translation initiation factor 2 (IF2mt) gene. The minimal promoter region for the human IF2mt gene contains binding sites for Nuclear Respiratory Factor 2 (NRF-2), which is often involved in the transcription of mitochondrial-related genes. Electrophoresis mobility shift assay (EMSA) analyses indicated that NRF-2 alpha/beta binds to the IF2mt promoter. Reporter assays, where HEK293T cells were co-transfected with an NRF-2 alpha/beta-expressing vector and/or an IF2mt promoter reporter vector, revealed that NRF-2 trans-activates the IF2mt promoter. NRF-2 sites were also found in the promoters of several other mitochondrial translation factors, which suggests NRF-2 may play a key role in the regulation of mitochondrial protein synthesis. (c) 2006 Elsevier B.V. and Mitochondria Research Society. All rights reserved.

    DOI

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    8
    被引用数
    (Scopus)
  • PURE technology for protein production

    Takuya Ueda

    SEIKAGAKU   79 ( 3 ) 205 - 212  2007年03月  [査読有り]

  • Chaperone properties of mammalian mitochondrial translation elongation factor Tu

    Hiroaki Suzuki, Takuya Ueda, Hideki Taguchi, Nono Takeuchi

    JOURNAL OF BIOLOGICAL CHEMISTRY   282 ( 6 ) 4076 - 4084  2007年02月  [査読有り]

     概要を見る

    The main function of the prokaryotic translation elongation factor Tu (EF-Tu) and its eukaryotic counterpart eEF1A is to deliver aminoacyl-tRNA to the A-site on the ribosome. In addition to this primary function, it has been reported that EF-Tu from various sources has chaperone activity. At present, little information is available about the chaperone activity of mitochondrial EF-Tu. In the present study, we have examined the chaperone function of mammalian mitochondrial EF-Tu (EF-Tumt). We demonstrate that recombinant EF-Tumt prevents thermal aggregation of proteins and enhances protein refolding in vitro and that this EF-Tumt chaperone activity proceeds in a GTP-independent manner. We also demonstrate that, under heat stress, the newly synthesized peptides from the mitochondrial ribosome specifically co-immunoprecipitate with EF-Tumt and are destabilized in EF-Tumt-overexpressing cells. We show that most of the EF-Tumt localizes on the mitochondrial inner membrane where most mitochondrial ribosomes are found. We discuss the possible role of EF-Tumt chaperone activity in protein quality control in mitochondria, with regard to the recently reported in vivo chaperone function of eEF1A.

    DOI

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    64
    被引用数
    (Scopus)
  • The PURE System: A Minimal Cell-Free Translation System

    Ying, B-W., Shimizu, Y., Ueda, T.

    Cell-Free ProteinExpression     76 - 83  2007年  [査読有り]

  • Efficient protein selection based on ribosome display system with purified components

    Hiroyuki Ohashi, Yoshihiro Shimizu, Bei-Wen Ying, Takuya Ueda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   352 ( 1 ) 270 - 276  2007年01月  [査読有り]

     概要を見る

    Using the PURE (Protein synthesis Using Recombinant Elements) system, we developed an efficient and highly controllable ribosome display method for selection of functional protein. The PURE system is composed of purified factors and enzymes that are responsible for gene expression in Escherichia coli. We performed the detailed analyses and optimization of the ribosome display system and demonstrated the formation of stable mRNA/ribosome/polypeptide ternary complexes. As complex formation is fundamental to successful ribosome display, these improvements resulted in a dramatic increase in the mRNA recovery rate. As a result, a similar to 12,000-fold enrichment of single-chain antibody (scFv) cDNA was achieved in a single round of selection. Specific selection of scFv mRNA from a 1:10(10) dilution in competitor mRNA was achieved with only three rounds of affinity selection. These findings, together with the results in the accompanying paper [T. Matsuura, H. Yanagida, J. Ushioda, I. Urabe, T. Yomo, Nascent chain, RNA, and ribosome complexes generated by pure translation system (see the accompanying paper).], demonstrate that the PURE system can provide a basis for reliable and reproducible ribosome display. (c) 2006 Elsevier Inc. All rights reserved.

    DOI

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    109
    被引用数
    (Scopus)
  • Kinetic analysis of the effects of translation enhancers in translation initiation.

    Takahashi, S., Furusawa, H., Shimizu, Y., Ueda, T., Okahata, Y.

    Nucleic acids symposium series (2004)   ( 51 ) 45 - 46  2007年  [査読有り]

  • Crystal structures of leucyl/phenylalanyl-tRNA-protein transferase and its complex with an aminoacyl-tRNA analog

    Kyoko Suto, Yoshihiro Shimizu, Kazunori Watanabe, Takuya Ueda, Shuya Fukai, Osamu Nureki, Kozo Tomita

    EMBO JOURNAL   25 ( 24 ) 5942 - 5950  2006年12月  [査読有り]

     概要を見る

    Eubacterial leucyl/phenylalanyl-tRNA protein transferase (L/F- transferase), encoded by the aat gene, conjugates leucine or phenylalanine to the N-terminal Arg or Lys residue of proteins, using Leu-tRNA Leu or Phe-tRNA Phe as a substrate. The resulting N-terminal Leu or Phe acts as a degradation signal for the ClpS-ClpAP-mediated N-end rule protein degradation pathway. Here, we present the crystal structures of Escherichia coli L/F-transferase and its complex with an aminoacyl-tRNA analog, puromycin. The C-terminal domain of L/F-transferase consists of the GCN5-related N-acetyltransferase fold, commonly observed in the acetyltransferase superfamily. The p-methoxybenzyl group of puromycin, corresponding to the side chain of Leu or Phe of Leu-tRNA(Leu) or Phe-tRNA(Phe), as accommodated in a highly hydrophobic pocket, with a shape and size suitable for hydrophobic amino-acid residues lacking a branched b-carbon, such as leucine and phenylalanine. Structure-based mutagenesis of L/F-transferase revealed its substrate specificity. Furthermore, we present a model of the L/F-transferase complex with tRNA and substrate proteins bearing an N-terminal Arg or Lys.

    DOI

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    54
    被引用数
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  • Cell-free translation systems for protein engineering

    Yoshihiro Shimizu, Yutetsu Kuruma, Bei-Wen Ying, So Umekage, Takuya Ueda

    FEBS JOURNAL   273 ( 18 ) 4133 - 4140  2006年09月  [査読有り]

     概要を見る

    Cell-free translation systems have developed significantly over the last two decades and improvements in yield have resulted in their use for protein production in the laboratory. These systems have protein engineering applications, such as the production of proteins containing unnatural amino acids and development of proteins exhibiting novel functions. Recently, it has been suggested that cell-free translation systems might be used as the fundamental basis for cell-like systems. We review recent progress in the field of cell-free translation systems and describe their use as tools for protein production and engineering.

    DOI

    Scopus

    82
    被引用数
    (Scopus)
  • Co-translational binding of GroEL to nascent polypeptides is followed by post-translational encapsulation by GroES to mediate protein folding

    Bei-Wen Ying, Hideki Taguchi, Takuya Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 31 ) 21813 - 21819  2006年08月  [査読有り]

     概要を見る

    The eubacterial chaperonins GroEL and GroES are essential chaperones and primarily assist protein folding in the cell. Although the molecular mechanism of the GroEL system has been examined previously, the mechanism by which GroEL and GroES assist folding of nascent polypeptides during translation is still poorly understood. We previously demonstrated a co-translational involvement of the Escherichia coli GroEL in folding of newly synthesized polypeptides using a reconstituted cell-free translation system ( Ying, B. W., Taguchi, H., Kondo, M., and Ueda, T. ( 2005) J. Biol. Chem. 280, 12035 - 12040). Employing the same system here, we further characterized the mechanism by which GroEL assists folding of translated proteins via encapsulation into the GroEL-GroES cavity. The stable co-translational association between GroEL and the newly synthesized polypeptide is dependent on the length of the nascent chain. Furthermore, GroES is capable of interacting with the GroEL-nascent peptide-ribosome complex, and experiments using a single-ring variant of GroEL clearly indicate that GroES association occurs only at the trans-ring, not the cis-ring, of GroEL. GroEL holds the nascent chain on the ribosome in a polypeptide length-dependent manner and post-translationally encapsulates the polypeptide using the GroES cap to accomplish the chaperonin-mediated folding process.

    DOI

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    32
    被引用数
    (Scopus)
  • SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans- translation initiation

    Y Shimizu, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   281 ( 23 ) 15987 - 15996  2006年06月  [査読有り]

     概要を見る

    Bacterial tmRNA rescues ribosomes that stall because of defective mRNAs via the trans-translation process. Although entry of the charged transfer messenger RNA ( tmRNA) into the ribosome proceeded in the absence of elongation factor (EF-Tu) and in the presence of EF-Tu and the antibiotic kirromycin, evidence was found for the involvement of EF-Tu in trans-translation initiation. The polyalanine synthesis system attained by using a tmRNA variant consisting of only the tRNA-like domain revealed that it was completely dependent on the presence of SmpB and greatly enhanced by EF-Tu and EF-G. Actually, ribosome-dependent GTPase activity of EF-Tu was stimulated by the addition of SmpB and tmRNA but independently of template mRNA, demonstrating that SmpB compensates for the lack of codon-anticodon interaction during the first step of the trans-translation initiation. Based on these results, we suggest that SmpB structurally mimics the anticodon arm of tRNA and elicits GTP hydrolysis of EF-Tu upon tmRNA accommodation in the A site of the ribosome.

    DOI

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    34
    被引用数
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  • The role of interface framework residues in determining antibody V-H/V-L interaction strength and antigen-binding affinity

    K Masuda, K Sakamoto, M Kojima, T Aburatani, T Ueda, H Ueda

    FEBS JOURNAL   273 ( 10 ) 2184 - 2194  2006年05月  [査読有り]

     概要を見る

    While many antibodies with strong antigen-binding affinity have stable variable regions with a strong antibody heavy chain variable region fragment (V-H)/antibody light chain variable region fragment (V-L) interaction, the anti-lysozyme IgG HyHEL-10 has a fairly strong affinity, yet a very weak V-H/V-L interaction strength, in the absence of antigen. To investigate the possible relationship between antigen-binding affinity and V-H/V-L interaction strength, a novel phage display system that can switch two display modes was employed. We focused on the two framework region 2 regions of the HyHEL-10 V-H and V-L, facing each other at the domain interface, and a combinatorial library was made in which each framework region 2 residue was mixed with that of D1.3, which has a far stronger V-H/V-L interaction. The phagemid library, encoding V-H gene 7 and V-L amber codon gene 9, was used to transform TG-1 (sup(+)), and the phages displaying functional variable regions were selected. The selected phages were then used to infect a nonsuppressing strain, and the culture supernatant containing V-H-displaying phages and soluble V-L fragment was used to evaluate the V-H/V-L interaction strength. The results clearly showed the existence of a key framework region 2 residue (H39) that strongly affects V-H/V-L interaction strength, and a marked positive correlation between the antigen-binding affinity and the V-H/V-L interaction, especially in the presence of a set of particular V-L residues. The effect of the H39 mutation on the wild-type variable region was also confirmed by a SPR biosensor as a several-fold increase in antigen-binding affinity owing to an increased association rate, while a slight decrease was observed for the single-chain variable region.

    DOI

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    29
    被引用数
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  • Spermidine inhibits transient and stable ribosome subunit dissociation

    S Umekage, T Ueda

    FEBS LETTERS   580 ( 5 ) 1222 - 1226  2006年02月  [査読有り]

     概要を見る

    Recent light-scattering experiments and sucrose density gradient centrifugational analyses suggested that the 70S ribosome undergoes RRF- and EF-G-triggered transient subunit dissociation that is followed by IF3-induced stable dissociation. However, the experimental conditions did not include the ubiquitous cellular polyamine spermidine, which is required for efficient translation. We found that when spermidine was present, the transient dissociation was inhibited. Moreover, the published experiments used ribosome concentrations that were far lower than the physiological concentration. We found that when spermidine and higher ribosome concentrations were included in the experimental conditions, only very limited stable subunit dissociation was observed. These results suggest that neither transient nor stable dissociation occurs under physiological conditions applied here. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

    DOI

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    18
    被引用数
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  • Kinetic analysis of ribosome binding process onto mRNA using a quartz-crystal microbalance.

    Takahashi, S., Akita, R., Furusawa, H., Shimizu, Y., Ueda, T., Okahata, Y.

    Nucleic acids symposium series (2004)   ( 50 ) 49 - 50  2006年  [査読有り]

    DOI

    Scopus

    4
    被引用数
    (Scopus)
  • Protein synthesis by pure translation systems

    Y Shimizu, T Kanamori, T Ueda

    METHODS   36 ( 3 ) 299 - 304  2005年07月  [査読有り]

     概要を見る

    We have developed a partially recombinant, cell-free, protein-synthesis system reconstituted solely from those essential elements of the Escherichia coli translation system, termed protein synthesis using recombinant elements (PURE). It provides higher reaction controllability in comparison to crude cell-free protein-synthesis systems for translation studies and biotechnology applications. The PURE system stands out among translation methods in that it provides not only a simple and unique "reverse" purification method of separating the synthesized protein from reaction mixture, but also that the system can be tailor-made according to individual protein requirements. In this paper, two new approaches to obtaining active proteins are described: the use of molecular chaperones, and modification of the reaction conditions. Several possible applications of the PURE system are also discussed. (c) 2005 Elsevier Inc. All rights reserved.

    DOI

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    305
    被引用数
    (Scopus)
  • Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

    Y Kuruma, K Nishiyama, Y Shimizu, M Muller, T Ueda

    BIOTECHNOLOGY PROGRESS   21 ( 4 ) 1243 - 1251  2005年07月  [査読有り]

     概要を見る

    By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.

    DOI

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    62
    被引用数
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  • Human mitochondrial mRNAs are stabilized with polyadenylation regulated by mitochondria-specific poly(A) polymerase and polynucleotide phosphorylase

    T Nagaike, T Suzuki, T Katoh, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 20 ) 19721 - 19727  2005年05月  [査読有り]

     概要を見る

    Mammalian mitochondrial (mt) mRNAs have short poly(A) tails at their 3' termini that are post-transcriptionally synthesized by mt poly(A) polymerase (PAP). The polyadenylation of mt mRNAs is known to be a key process needed to create UAA stop codons that are not encoded in mtDNA. In some cases, polyadenylation is required for the tRNA maturation by editing of its 3' terminus. However, little is known about the functional roles the poly(A) tail of mt mRNAs plays in mt translation and RNA turnover. Here we show human mt PAP (hmtPAP) and human polynucleotide phosphorylase (hPNPase) control poly(A) synthesis in human mitochondria. Partial inactivation of hmtPAP by RNA interference using small interfering RNA in HeLa cells resulted in shortened poly(A) tails and decreased steady state levels of some mt mRNAs as well as their translational products. Moreover, knocking down hmtPAP generated markedly defective mt membrane potentials and reduced oxygen consumption. In contrast, knocking down hPNPase showed significantly extended poly(A) tails of mt mRNAs. These results demonstrate that the poly(A) length of human mt mRNAs is controlled by polyadenylation by hmtPAP and deadenylation by hPNPase, and polyadenylation is required for the stability of mt mRNAs.

    DOI

  • Esterification of Eschericia coli tRNAs with D-histidine and D-lysine by aminoacyl-tRNA synthetases

    T Takayama, T Ogawa, M Hidaka, Y Shimizu, T Ueda, H Masaki

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   69 ( 5 ) 1040 - 1041  2005年05月  [査読有り]

     概要を見る

    It is generally believed that only L-amino acids are acceptable in protein synthesis, though some D-amino acids, including D-tyrosine, D-aspartate, and D-tryptophan are known to be bound enzymatically to tRNAs. In this report, we newly show that D-histidine and D-lysine are also able to be the substrates of respective Escherichia coli aminoacyl-tRNA synthetases.

    DOI

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    15
    被引用数
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  • Human mitochondrial mRNAs are stabilized with polyadenylation regulated by mitochondria-specific poly(A) polymerase and polynucleotide phosphorylase

    T Nagaike, T Suzuki, T Katoh, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 20 ) 19721 - 19727  2005年05月  [査読有り]

     概要を見る

    Mammalian mitochondrial (mt) mRNAs have short poly(A) tails at their 3' termini that are post-transcriptionally synthesized by mt poly(A) polymerase (PAP). The polyadenylation of mt mRNAs is known to be a key process needed to create UAA stop codons that are not encoded in mtDNA. In some cases, polyadenylation is required for the tRNA maturation by editing of its 3' terminus. However, little is known about the functional roles the poly(A) tail of mt mRNAs plays in mt translation and RNA turnover. Here we show human mt PAP (hmtPAP) and human polynucleotide phosphorylase (hPNPase) control poly(A) synthesis in human mitochondria. Partial inactivation of hmtPAP by RNA interference using small interfering RNA in HeLa cells resulted in shortened poly(A) tails and decreased steady state levels of some mt mRNAs as well as their translational products. Moreover, knocking down hmtPAP generated markedly defective mt membrane potentials and reduced oxygen consumption. In contrast, knocking down hPNPase showed significantly extended poly(A) tails of mt mRNAs. These results demonstrate that the poly(A) length of human mt mRNAs is controlled by polyadenylation by hmtPAP and deadenylation by hPNPase, and polyadenylation is required for the stability of mt mRNAs.

    DOI

  • Co-translational involvement of the chaperonin GroEL in the folding of newly translated polypeptides

    BW Ying, H Taguchi, M Kondo, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 12 ) 12035 - 12040  2005年03月  [査読有り]

     概要を見る

    A large fraction of the newly translated polypeptides emerging from the ribosome require certain proteins, the so-called molecular chaperones, to assist in their folding. In Escherichia coli, three major chaperone systems are considered to contribute to the folding of newly synthesized cytosolic polypeptides. Trigger factor (TF), a ribosome-tethered chaperone, and DnaK are known to exhibit overlapping co-translational roles, whereas the cage-shaped GroEL, with the aid of the co-chaperonin, GroES, and ATP, is believed to be implicated in folding only after the polypeptides are released from the ribosome. However, the recent finding that GroEL-GroES overproduction permits the growth of E. coli cells lacking both TF and DnaK raised questions regarding the separate roles of these chaperones. Here, we report the puromycin-sensitive association of GroEL-GroES with translating ribosomes in vivo. Further experiments in vitro, using a reconstituted cell-free translation system, clearly demonstrate that GroEL associates with the translation complex and accomplishes proper folding by encapsulating the newly translated polypeptides in the central cavity formed by GroES. Therefore, we propose that GroEL is a versatile chaperone, which participates in the folding pathway co-translationally and also achieves correct folding post-translationally.

    DOI

    Scopus

    55
    被引用数
    (Scopus)
  • Structural basis for template-independent RNA polymerization

    K Tomita, S Fukai, R Ishitani, T Ueda, N Takeuchi, DG Vassylyev, O Nureki

    NATURE   430 ( 7000 ) 700 - 704  2004年08月  [査読有り]

     概要を見る

    The 3'-terminal CCA nucleotide sequence ( positions 74 - 76) of transfer RNA is essential for amino acid attachment(1) and interaction with the ribosome(2-4) during protein synthesis. The CCA sequence is synthesized de novo and/or repaired by a template-independent RNA polymerase, 'CCA-adding enzyme', using CTP and ATP as substrates(5). Despite structural and biochemical studies(5-8), the mechanism by which the CCA-adding enzyme synthesizes the defined sequence without a nucleic acid template remains elusive. Here we present the crystal structure of Aquifex aeolicus CCA-adding enzyme, bound to a primer tRNA lacking the terminal adenosine and an incoming ATP analogue, at 2.8 Angstrom resolution. The enzyme enfolds the acceptor T helix of the tRNA molecule. In the catalytic pocket, C75 is adjacent to ATP, and their base moieties are stacked. The complementary pocket for recognizing C74-C75 of tRNA forms a 'protein template' for the penultimate two nucleotides, mimicking the nucleotide template used by template-dependent polymerases. These results are supported by systematic analyses of mutants. Our structure represents the 'pre-insertion' stage of selecting the incoming nucleotide and provides the structural basis for the mechanism underlying template-independent RNA polymerization.

    DOI

    Scopus

    91
    被引用数
    (Scopus)
  • Chaperone-assisted folding of a single-chain antibody in a reconstituted translation system

    BW Ying, H Taguchi, H Ueda, T Ueda

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   320 ( 4 ) 1359 - 1364  2004年08月  [査読有り]

     概要を見る

    A protein-synthesizing system based on a minimal set of purified components was used to investigate the roles molecular chaperones play in the folding of newly synthesized polypeptides. After we ascertained that this system lacks intrinsic chaperones, the effect of adding chaperones in a co-translational or post-translational manner was directly evaluated. An aggregation-prone single-chain antibody was used as the model nascent chain. The participation of the trigger factor or the DnaK system during translation efficiently increased the level of functional protein that was generated. In addition, both systems also acted as chaperones after translation had been stopped. In contrast, the GroEL/ES system showed little or no co- or post-translational assistance in folding. (C) 2004 Published by Elsevier Inc.

    DOI

    Scopus

    41
    被引用数
    (Scopus)
  • Recombinant antigen-based immuno-slot blot method for serodiagnosis of syphilis

    NS Sato, T Suzuki, T Ueda, K Watanabe, RDC Hirata, MH Hirata

    BRAZILIAN JOURNAL OF MEDICAL AND BIOLOGICAL RESEARCH   37 ( 7 ) 949 - 955  2004年07月  [査読有り]

     概要を見る

    Three recombinant antigens of Treponema pallidum Nichols strain were fused with GST, cloned and expressed in Escherichia coli, resulting in high levels of GST-rTp47 and GST-rTp17 expression, and supplementation with arginine tRNA for the AGR codon was needed to obtain GST-rTp15 overexpression. Purified fusion protein yields were 1.9, 1.7 and 5.3 mg/l of cell culture for GST-rTp47, GST-rTp17 and GST-rTp15, respectively. The identities of the antigens obtained were confirmed by automated DNA sequencing using ABI Prism 310 and peptide mapping by Finningan LC/MS. These recombinant antigens were evaluated by immuno-slot blot techniques applied to 137 serum samples from patients with a clinical and laboratory diagnosis of syphilis (61 samples), from healthy blood donors (50 samples), individuals with sexually transmitted disease other than syphilis (3 samples), and from individuals with other spirochetal diseases such as Lyme disease (20 samples) and leptospirosis (3 samples). The assay had sensitivity of 95.1% (95% Cl, 86.1 to 98.7%) and a specificity of 94.7% (95% Cl, 87.0 to 98.7%); a stronger reactivity was observed with fraction rTp17. The immunoreactivity results showed that fusion recombinant antigens based-immuno-slot blot techniques are suitable for use in diagnostic assays for syphilis.

    DOI

    Scopus

    8
    被引用数
    (Scopus)
  • Synthesis of disulfide bond containing protein using PURESYSTEM and its exploitation as an oxidoreductase-free model

    Hidefumi Kuwata, Sachiko Takami, Takashi Kanamori, Yoshihiro Shimizu, Go Hattori, Koji Sato, Yoshinori Saruta, Yukie Sameshima, Tomoko Miyagi, Bei-Wen Ying, Takuya Ueda

    CELL STRUCTURE AND FUNCTION   29   93 - 93  2004年05月  [査読有り]

  • The pathogenic A4269G mutation in human mitochondrial tRNA(Ile) alters the T-stem structure and decreases the binding affinity for elongation factor Tu

    N Hino, T Suzuki, T Yasukawa, K Seio, K Watanabe, T Ueda

    GENES TO CELLS   9 ( 3 ) 243 - 252  2004年03月  [査読有り]

     概要を見る

    The A4269G mutation in the human mitochondrial (mt) tRNA(Ile) gene is associated with fatal cardiomyopathy. This mutation completely inhibits protein synthesis in mitochondria, thereby significantly reducing their respiratory activity. The steady-state amount of tRNA(Ile) in cells bearing the A4269G mutation is almost half that of control cells. We previously reported that this mutation causes tRNA(Ile) to be unstable both in vivo and in vitro. To investigate whether the instability of the mutant tRNA(Ile) is due to structural alterations, a nuclease-probing experiment was performed with a mitochondrial enzymatic extract, which showed that the A4269G mutation destabilizes the T-stem of the mutant tRNA(Ile). In addition, measurements of the binding affinity of the aminoacylated mutant tRNA(Ile) for mt elongation factor Tu (EF-Tu) showed that the mutant tRNA(Ile) binds mt EF-Tu less efficiently than the wild-type does. This observation provides insight into the molecular pathology associated with tRNA dysfunction caused by this pathogenic point mutation.

    DOI

    Scopus

    17
    被引用数
    (Scopus)
  • Evidence for the translation initiation of leaderless mRNAs by the intact 70 S ribosome without its dissociation into subunits in eubacteria

    T Udagawa, Y Shimizu, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   279 ( 10 ) 8539 - 8546  2004年03月  [査読有り]

     概要を見る

    In eubacteria, the dissociation of the 70 S ribosome into the 30 S and 50 S subunits is the essential first step for the translation initiation of canonical mRNAs that possess 5'-leader sequences. However, a number of leaderless mRNAs that start with the initiation codon have been identified in some eubacteria. These have been shown to be translated efficiently in vivo. Here we investigated the process by which leaderless mRNA translation is initiated by using a highly reconstituted cell-free translation system from Escherichia coli. We found that leaderless mRNAs bind preferentially to 70 S ribosomes and that the leaderless mRNA . 70 S . fMet-tRNA complex can transit from the initiation to the elongation phase even in the absence of initiation factors ( IFs). Moreover, leaderless mRNA translation proceeds more efficiently if the intact 70 S ribosome is involved compared with the 30 S subunit. Furthermore, excess amounts of IF3 inhibit leaderless mRNA translation, probably because it promotes the disassembly of the 70 S ribosome into subunits. Finally, excess amounts of fMet-tRNA facilitate the IF-independent translation of leaderless mRNA. These observations strongly suggest that leaderless mRNA translation is initiated by the assembled 70 S ribosome and thereby bypasses the dissociation process.

    DOI

    Scopus

    94
    被引用数
    (Scopus)
  • [Reconstitution of in vitro protein maturation system: the pure approach for protein maturation]

    Ying, B. W., Ueda, T.

    Tanpakushitsu Kakusan Koso   49 ( 7 Suppl ) 834 - 7  2004年  [査読有り]

  • [In vitro protein synthesis system: PURESYSTEM--completely reconstituted system derived from Escherichia coli]

    Shimizu, Y., Ueda, T.

    Tanpakushitsu Kakusan Koso   49 ( 11 Suppl ) 1520 - 6  2004年  [査読有り]

  • [Cell-free gene expression system]

    Ueda, T.

    Seikagaku   76 ( 2 ) 130 - 4  2004年  [査読有り]

  • Down-regulation of the mitochondrial translation system during terminal differentiation of HL-60 cells by 12-O-tetradecanoyl-1-phorbol-13-acetate - Comparison with the cytoplasmic translation system

    N Takeuchi, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 46 ) 45318 - 45324  2003年11月  [査読有り]

     概要を見る

    Mitochondrial (mt) biogenesis depends on both the nuclear and mt genomes, and a coordination of these two genetic systems is necessary for proper cell functioning. Little is known about the regulatory mechanisms of mt translation or about the expression of mt translation factors. Here, we studied the expression of mt translation factors during 12-O-tetradecanoyl-1-phorbol-13-acetate (TPA)-induced terminal differentiation of HL-60 cells. For all mt translation factors investigated, mRNA expression was markedly downregulated in a coordinate and specific manner, whereas mRNA levels for the cytoplasmic translation factors showed only a slight reduction. An actinomycin D chase study and nuclear run-on assay revealed that the TPA-induced decrease in mt elongation factor Tu (EF-Tumt) mRNA mainly results from decreased mRNA stability. Polysome analysis showed that there was no significant translational control of mt translation factor (EF-Tumt, ribosomal proteins L7/L12mt and S12mt) mRNA expression during differentiation. Thus, the decreased protein level of one of these mt translation factors (EF-Tumt) simply reflects its decreased mRNA level. It was also demonstrated by pulse labeling of mt translation products that the down-regulation of mt translational activity is actually associated with down-regulated mt translation factor expression during cellular differentiation. Our results illustrate that the regulatory mechanisms of mt translational activity upon terminal differentiation ( in response to the growth arrest) is different to that of the cytoplasmic system, where the control of mRNA translational efficiency of major translation factors is the central mechanism for their down-regulation.

    DOI

    Scopus

    14
    被引用数
    (Scopus)
  • Decreased CCA-addition in human mitochondrial tRNAs bearing a pathogenic A4317G or A10044G mutation

    Y Tomari, N Hino, T Nagaike, T Suzuki, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   278 ( 19 ) 16828 - 16833  2003年05月  [査読有り]

     概要を見る

    Pathogenic point mutations in mitochondrial tRNA genes are known to cause a variety of human mitochondrial diseases. Reports have associated an A4317G mutation in the mitochondrial tRNAIle gene with fatal infantile cardiomyopathy and an A10044G mutation in the mitochondrial tRNAGly gene with sudden infant death syndrome. Here we demonstrate that both mutations inhibit in vitro CCA-addition to the respective tRNA by the human mitochondrial CCA-adding enzyme. Structures of these two mutant tRNAs were examined by nuclease probing. In the case of the A4317G tRNAIle mutant, structural rearrangement of the T-arm region, conferring an aberrantly stable T-arm structure and an increased T-m value, was clearly observed. In the case of the A10044G tRNAGly mutant, high nuclease sensitivity in both the T- and D-loops suggested a weakened interaction between the loops. These are the first reported instances of inefficient CCA-addition being one of the apparent molecular pathogeneses caused by pathogenic point mutations in human mitochondrial tRNA genes.

    DOI

    Scopus

    30
    被引用数
    (Scopus)
  • A point mutation in ribosomal protein L7/L12 reduces its ability to form a compact dimer structure and to assemble into the GTPase center

    T Nomura, R Mochizuki, ER Dabbs, Y Shimizu, T Ueda, A Hachimori, T Uchiumi

    BIOCHEMISTRY   42 ( 16 ) 4691 - 4698  2003年04月  [査読有り]

     概要を見る

    An Escherichia coli mutant, LL103, harboring a mutation (Ser15 to Phe) in ribosomal protein L7/L12 was isolated among revertants of a streptomycin-dependent strain. In the crystal structure of the L7/L12 dimer, residue 15 within the N-terminal domain contacts the C-terminal domain of the partner monomer. We tested effects of the mutation on molecular assembly by biochemical approaches. Gel electrophoretic analysis showed that the Phe15-L7/L12 variant had reduced ability in binding to L10, an effect enhanced in the presence of 0.05% of nonionic detergent. Mobility of Phe15-L7/L12 on gel containing the detergent was very low compared to the wild-type proteins, presumably because of an extended structural state of the mutant L7/L12. Ribosomes isolated from LL103 cells contained a reduced amount of L7/L12 and showed low levels (15-30% of wild-type ribosomes) of activities dependent on elongation factors and in translation of natural mRNA. The ribosomal activity was completely recovered by addition of an excess amount of Phe15-L7/L12 to the ribosomes, suggesting that the mutant L7/L12 exerts normal functions when bound on the ribosome. The interaction of Ser15 with the C-terminal domain of the partner molecule seems to contribute to formation of the compact dimer structure and its efficient assembly into the ribosomal GTPase center. We propose a model relating compact and elongated forms of L7/L12 dimers. Phe15-L7/L12 provides a new tool for studying the functional structure of the homodimer.

    DOI

    Scopus

    9
    被引用数
    (Scopus)
  • A novel screening system for self-mRNA targeting proteins

    BW Ying, T Suzuki, Y Shimizu, T Ueda

    JOURNAL OF BIOCHEMISTRY   133 ( 4 ) 485 - 491  2003年04月  [査読有り]

     概要を見る

    Here we describe the application of an in vitro translation system for genetic screening, to identify RNA-binding proteins that bind to their own mRNAs. It is a relatively novel system designed using an advanced cell-free translation system reconstructed with purified translational components. Due to the absence of nucleases and proteases, the complex of mRNA and nascent polypeptide synthesized in this system is expected to exhibit high stability ensuring the following efficient selection toward the protein. Escherichia coli ribosomal protein S15, which is known to bind to its own mRNA, was employed as a model molecule to evaluate the system. Wild-type S15 mRNA specifically isolated from a mutant mRNA lacking the secondary structure responsible for binding the S15 protein accumulated markedly after several rounds of selection-amplification. The success of this selection demonstrates the potentiality of the systematic screening of self-mRNA targeting proteins through direct and functional selection. This strategy as a method to identify peptides or proteins that bind to their own mRNAs, is of general interest and has different potential applications, such as, the identification of new regulatory proteins or peptide motifs for RNA recognition, the study of self-mRNA-protein interactions, etc.

    DOI

    Scopus

    8
    被引用数
    (Scopus)
  • The cephalopod Loligo bleekeri mitochondrial genome: Multiplied noncoding regions and transposition of tRNA genes

    K Tomita, S Yokobori, T Oshima, T Ueda, K Watanabe

    JOURNAL OF MOLECULAR EVOLUTION   54 ( 4 ) 486 - 500  2002年04月  [査読有り]

     概要を見る

    We previously reported the sequence of a 9260-bp fragment of mitochondrial (mt) DNA of the cephalopod Loligo bleekeri [J. Sasuga et al. (1999) J. Mol. Evol. 48:692-702]. To clarify further the characteristics of Loligo mtDNA. we have sequenced an 8148-bp fragment to reveal the complete mt genome sequence. Loligo mtDNA is 17,211 bp long and possesses a standard set of metazoan mt genes. Its gene arrangement is not identical to any other metazoan mt gene arrangement reported so far. Three of the 19 noncoding regions longer than 10 bp are 515, 507, and 509 bp long, and their sequences are nearly identical, suggesting that multiplication of these noncoding regions occurred in an ancestral Loligo mt genome. Comparison of the gene arrangements of Loligo, Katharina tunicata. and Littorina saxatilis mt genomes revealed that 17 tRNA genes of the Loligo mt genome are adjacent to noncoding regions. A majority (15 tRNA genes) of their counterparts is found in two tRNA gene clusters of the Katharina mt genome. Therefore, the Loligo mt genome (17 tRNA genes) may have spread over the genome, and this may have been coupled with the multiplication of the noncoding regions. Maximum likelihood analysis of mt protein genes supports the clade Mollusca + Annelida + Brachiopoda but fails to infer the relationships among Katharina, Loligo, and three gastropod species.

    DOI

    Scopus

    63
    被引用数
    (Scopus)
  • The role of SmpB protein in trans-translation

    Y Shimizu, T Ueda

    FEBS LETTERS   514 ( 1 ) 74 - 77  2002年03月  [査読有り]

     概要を見る

    The function of SmpB protein in the trans-translation system was evaluated using the well-defined cell-free translation system consisting of purified ribosome, alanyl-tRNA synthetase and elongation factors. The analysis showed that SmpB protein enhances alanine-accepting activity of tmRNA and that SmpB protein and tmRNA are sufficient to complete the transtranslation process in the presence of translational components. Moreover, SmpB is indispensable in the addition of tag-peptide onto ribosomes by tmRNA. In particular, the A-site binding of tmRNA is inhibited in the absence of SmpB. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

    DOI

    Scopus

    58
    被引用数
    (Scopus)
  • tRNA recognition by CCA-adding enzyme.

    Tomari Y, Suzuki T, Ueda T

    Nucleic acids research. Supplement (2001)   ( 2 ) 77 - 78  2002年  [査読有り]  [国際誌]

    PubMed

  • Identification and characterization of mammalian mitochondrial tRNA nucleotidyltransferases

    T Nagaike, T Suzuki, Y Tomari, C Takemoto-Hori, F Negayama, K Watanabe, T Ueda

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 43 ) 40041 - 40049  2001年10月  [査読有り]

     概要を見る

    The CCA-adding enzyme (ATP:tRNA adenylyltransferase or CTP:tRNA cytidylyltransferase (EC 2.7.7.25)) generates the conserved CCA sequence responsible for the attachment of amino acid at the 3&apos; terminus of tRNA molecules. It was shown that enzymes from various organisms strictly recognize the elbow region of tRNA formed by the conserved D- and T-loops. However, most of the mammalian mitochondrial (mt) tRNAs lack consensus sequences in both D- and T-loops. To characterize the mammalian mt CCA-adding enzymes, we have partially purified the enzyme from bovine liver mitochondria and determined cDNA sequences from human and mouse dbESTs by mass spectrometric analysis. The identified sequences contained typical amino-terminal peptides for mitochondrial protein import and had characteristics of the class II nucleotidyltransferase superfamily that includes eukaryotic and eubacterial CCA-adding enzymes. The human recombinant enzyme was overexpressed in Escherichia coli, and its CCA-adding activity was characterized using several int tRNAs as substrates. The results clearly show that the human mt CCA-adding enzyme can efficiently repair mt tRNAs that are poor substrates for the E. coli enzyme although both enzymes work equally well on cytoplasmic tRNAs. This suggests that the mammalian mt enzymes have evolved so as to recognize mt tRNAs with unusual structures.

    DOI

    Scopus

    91
    被引用数
    (Scopus)
  • Proteomic analysis of the mammalian mitochondrial ribosome - Identification of protein components in the 28 S small subunit

    T Suzuki, M Terasaki, C Takemoto-Hori, T Hanada, T Ueda, A Wada, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 35 ) 33181 - 33195  2001年08月  [査読有り]

     概要を見る

    The mammalian mitochondrial ribosome (mitoribosome) has a highly protein-rich composition with a small sedimentation coefficient of 55 S, consisting of 39 S large and 28 S small subunits. In the previous study, we analyzed 39 S large subunit proteins from bovine mitoribosome (Suzuki, T., Terasaki, M, Takemoto-Hori, C., Hanada, T., Ueda, T., Wada, A., and Watanabe, VL (2001) J. BioL Chem. 276,21724-21736). The results suggested structural compensation for the rRNA deficit through proteins of increased molecular mass in the mitoribosome. We report here the identification of 28 S small subunit proteins. Each protein was separated by radical-free high-reducing two-dimensional polyacrylamide gel electrophoresis and analyzed by liquid chromatography/mass spectrometry/mass spectrometry using electrospray ionization/ion trap mass spectrometer to identify DNA sequence by expressed sequence tag data base searches in silico. Twenty one proteins from the small subunit were identified, including 11 new proteins along with their complete cDNA sequences from human and mouse. In addition to these proteins, three new proteins were also identified in the 55 S mitoribosome. We have clearly identified a mitochondrial homologue of S12, which is a key regulatory protein of translation fidelity and a candidate for the autosomal dominant deafness gene, DFNA4. The apoptosis-related protein DAP3 was found to be a component of the small subunit, indicating a new function for the mitoribosome in programmed cell death. In summary, we have mapped a total of 55 proteins from the 55 S mitoribosome on the two-dimensional polyacrylamide gels.

    DOI

    Scopus

    129
    被引用数
    (Scopus)
  • S3F01試験管内遺伝子発現系の構築

    上田 卓也

    生物物理   41   S22  2001年

    DOI CiNii

  • Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation

    T Yasukawa, T Suzuki, N Ishii, T Ueda, S Ohta, K Watanabe

    FEBS LETTERS   467 ( 2-3 ) 175 - 178  2000年02月  [査読有り]

     概要を見る

    A mitochondrial tRNA(Lys) gene mutation at nucleotide position 8344 is responsible for the myoclonus epilepsy associated with ragged-red fibers (MERRF) subgroup of mitochondrial encephalomyopathies. Here, we show that normally modified uridine at the anticodon wobble position remains unmodified in the purified mutant tRNA(Lys). We hale reported a similar modification defect at the same position in two mutant mitochondrial tRNAs(Leu)(UUR) in another subgroup, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), indicating this defect is common in the two hinds of tRNA molecules with the respective mutations of the two major mitochondrial encephalomyopathies. We therefore suggest the defect in the anticodon is responsible, through the translational process, for the pathogenesis of mitochondrial diseases. (C) 2000 Federation of European Biochemical Societies.

    DOI DOI2

    Scopus

    117
    被引用数
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  • Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation

    T Yasukawa, T Suzuki, N Ishii, T Ueda, S Ohta, K Watanabe

    FEBS LETTERS   467 ( 2-3 ) 175 - 178  2000年02月  [査読有り]

     概要を見る

    A mitochondrial tRNA(Lys) gene mutation at nucleotide position 8344 is responsible for the myoclonus epilepsy associated with ragged-red fibers (MERRF) subgroup of mitochondrial encephalomyopathies. Here, we show that normally modified uridine at the anticodon wobble position remains unmodified in the purified mutant tRNA(Lys). We hale reported a similar modification defect at the same position in two mutant mitochondrial tRNAs(Leu)(UUR) in another subgroup, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), indicating this defect is common in the two hinds of tRNA molecules with the respective mutations of the two major mitochondrial encephalomyopathies. We therefore suggest the defect in the anticodon is responsible, through the translational process, for the pathogenesis of mitochondrial diseases. (C) 2000 Federation of European Biochemical Societies.

    DOI

  • Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretxi (Chordata, Urochordata)

    S Yokobori, T Ueda, G Feldmaier-Fuchs, S Paabo, R Ueshima, A Kondow, K Nishikawa, K Watanabe

    GENETICS   153 ( 4 ) 1851 - 1862  1999年12月  [査読有り]

     概要を見る

    The complete nucleotide sequence of the 14,771-bp-long mitochondrial (rnt) DNA of a urochordate (Chordata)-the ascidian Halocynthia: roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(CAT)(Met) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(GCU)(Ser), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.

  • Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretxi (Chordata, Urochordata)

    S Yokobori, T Ueda, G Feldmaier-Fuchs, S Paabo, R Ueshima, A Kondow, K Nishikawa, K Watanabe

    GENETICS   153 ( 4 ) 1851 - 1862  1999年12月  [査読有り]

     概要を見る

    The complete nucleotide sequence of the 14,771-bp-long mitochondrial (rnt) DNA of a urochordate (Chordata)-the ascidian Halocynthia: roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(CAT)(Met) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(GCU)(Ser), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.

  • Peptide bond formation: Retraction

    Nitta, I, Y Kamada, H Noda, T Ueda, K Watanabe

    SCIENCE   283 ( 5410 ) 2019 - 2020  1999年03月  [査読有り]

  • Nucleotide sequences of animal mitochondrial tRNAs(Met) possibly recognizing both AUG and AUA codons.

    Takemoto, C., Ueda, T., Miura, K., Watanabe, K.

    Nucleic acids symposium series   ( 42 ) 77 - 78  1999年  [査読有り]

  • Erratum: (RNA (March 1998))

    I. Nitta, T. Ueda, K. Watanabe

    RNA   5 ( 5 ) 707  1999年  [査読有り]

    DOI PubMed

    Scopus

    4
    被引用数
    (Scopus)
  • 7-Methylguanosine at the anticodon wobble position of squid mitochondrial tRNA(Ser)GCU: molecular basis for assignment of AGA/AGG codons as serine in invertebrate mitochondria

    K Tomita, T Ueda, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1399 ( 1 ) 78 - 82  1998年07月  [査読有り]

     概要を見る

    In mitochondria of the squid, Loligo bleekeri, both the AGA and AGG codons are considered to correspond to serine instead of arginine as in the universal genetic code, and its genome encodes a single tRNA(Ser) gene with the anticodon GCT. Therefore, this gene product, tRNA(Ser)GCU, should be able to translate all four AGN (N; U, C, A, and G) codons as serine. To elucidate this recognition mechanism, the tRNA(Ser)GCU was isolated from squid liver and its complete nucleotide sequence determined. The tRNA(Ser)GCU was found to possess 7-methylguanosine (m(7)G) at the wobble position of the anticodon. This suggests that in the squid mitochondrial system, tRNA(Ser)GCU with the anticodon m(7)GCU can recognize not only the usual serine codons AGU and AGC, but also the unusual serine codons AGA and AGG, as in the case of starfish mitochondria (Matsuyama et al., J. Biol. Chem. 273 (1985) 3363-3368). (C) 1998 Elsevier Science B.V. All rights reserved.

    DOI DOI2

    Scopus

    38
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    (Scopus)
  • 7-Methylguanosine at the anticodon wobble position of squid mitochondrial tRNA(Ser)GCU: molecular basis for assignment of AGA/AGG codons as serine in invertebrate mitochondria

    K Tomita, T Ueda, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1399 ( 1 ) 78 - 82  1998年07月  [査読有り]

     概要を見る

    In mitochondria of the squid, Loligo bleekeri, both the AGA and AGG codons are considered to correspond to serine instead of arginine as in the universal genetic code, and its genome encodes a single tRNA(Ser) gene with the anticodon GCT. Therefore, this gene product, tRNA(Ser)GCU, should be able to translate all four AGN (N; U, C, A, and G) codons as serine. To elucidate this recognition mechanism, the tRNA(Ser)GCU was isolated from squid liver and its complete nucleotide sequence determined. The tRNA(Ser)GCU was found to possess 7-methylguanosine (m(7)G) at the wobble position of the anticodon. This suggests that in the squid mitochondrial system, tRNA(Ser)GCU with the anticodon m(7)GCU can recognize not only the usual serine codons AGU and AGC, but also the unusual serine codons AGA and AGG, as in the case of starfish mitochondria (Matsuyama et al., J. Biol. Chem. 273 (1985) 3363-3368). (C) 1998 Elsevier Science B.V. All rights reserved.

    DOI

  • Ascidian mitochondrial tRNA(Met) possessing unique structural characteristics

    A Kondow, S Yokobori, T Ueda, K Watanabe

    NUCLEOSIDES & NUCLEOTIDES   17 ( 1-3 ) 531 - 539  1998年01月  [査読有り]

     概要を見る

    Methionine tRNA was purified from muscle mitochondria of the ascidian Halocynthia roretzi and its RNA sequence was determined. Analysis of the nucleotide sequence revealed that unlike most metazoan mitochondrial tRNAs(Met), which have a highly conserved cytidine (C) or C-derivative at the wobble position, the H. roretzi mitochondrial tRNA(Met) possesses 5-carboxymethylaminomethyluridine (cmnm(5)U) at the first position of the anticodon. This is the first report of a single mitochondrial tRNA(Met) species having uridine (U) or a U-derivative at the wobble position.

    DOI

    Scopus

    12
    被引用数
    (Scopus)
  • The non-standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation?

    MAS Santos, T Ueda, K Watanabe, MF Tuite

    MOLECULAR MICROBIOLOGY   26 ( 3 ) 423 - 431  1997年11月  [査読有り]

     概要を見る

    A number of yeasts of the genus Candida translate the standard leucine-CUG codon as serine. This unique genetic code change is the only known alteration to the universal genetic code in cytoplasmic mRNAs, of either eukaryotes or prokaryotes, which involves reassignment of a sense codon. Translation of CUG as serine in these species is mediated by a novel serine-tRNA (ser-tRNA(CAG)), which uniquely has a guanosine at position 33, 5' to the anticodon, a position that is almost invariably occupied by a pyrimidine (uridine in general) in all other tRNAs. We propose that G-33 has two important functions: lowering the decoding efficiency of the ser-tRNA(CAG) and preventing binding of the leucyl-tRNA synthetase. This implicates this nucleotide as a key player in the evolutionary reassignment of the CUG codon. In addition, the novel ser-tRNA(CAG) has 1-methylguanosine (m(1)G-37) at position 37, 3' to the anticodon, which is characteristic of leucine, but not serine tRNAs. Remarkably, m(1)G-37 causes leucylation of the ser-tRNA(CAG) both in vitro and in vivo, making the CUG codon an ambiguous codon: the polysemous codon. This indicates that some Candida species tolerate ambiguous decoding and suggests either that (i) the genetic code change has not yet been fully established and is evolving at different rates in different Candida species; or (ii) CUG ambiguity is advantageous and represents the final stage of the reassignment. We propose that such dual specificity indicates that reassignment of the CUG codon evolved through a mechanism that required codon ambiguity and that ambiguous decoding evolved to generate genetic diversity and allow for rapid adaptation to environmental challenges.

  • cDNA sequence of a translational elongation factor Ts homologue from Caenorhabditis elegans: Mitochondrial factor-specific features found in the nematode homologue peptide

    Y Watanabe, K Kita, T Ueda, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1353 ( 1 ) 7 - 12  1997年07月  [査読有り]

     概要を見る

    The cDNA for a homologue of elongation factor Ts which probably functions in mitochondria has been sequenced from a nematode Caenorhabditis elegans. The deduced amino acid sequence (316 amino acids long) has a possible transit peptide sequence at the amino terminus and several common specific features for mammalian mitochondrial EF-Ts. The amino acid identities in the protein from C. elegans compared with those of bovine mitochondria and Eschericia coli are 29.5% and 24.0%, respectively. The C. elegans sequence was classified as a long EF-Ts (ca. 280 amino acids long) similar to peptides from mammalian mitochondria and eubacteria other than Thermus and cyanobacteria (except Spirulina platensis), rather than short EF-Ts (ca. 200 amino acids long) as those of Thermus, cyanobacteria and plastids. (C) 1997 Elsevier Science B.V.

    DOI DOI2

    Scopus

    2
    被引用数
    (Scopus)
  • cDNA sequence of a translational elongation factor Ts homologue from Caenorhabditis elegans: Mitochondrial factor-specific features found in the nematode homologue peptide

    Y Watanabe, K Kita, T Ueda, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1353 ( 1 ) 7 - 12  1997年07月  [査読有り]

     概要を見る

    The cDNA for a homologue of elongation factor Ts which probably functions in mitochondria has been sequenced from a nematode Caenorhabditis elegans. The deduced amino acid sequence (316 amino acids long) has a possible transit peptide sequence at the amino terminus and several common specific features for mammalian mitochondrial EF-Ts. The amino acid identities in the protein from C. elegans compared with those of bovine mitochondria and Eschericia coli are 29.5% and 24.0%, respectively. The C. elegans sequence was classified as a long EF-Ts (ca. 280 amino acids long) similar to peptides from mammalian mitochondria and eubacteria other than Thermus and cyanobacteria (except Spirulina platensis), rather than short EF-Ts (ca. 200 amino acids long) as those of Thermus, cyanobacteria and plastids. (C) 1997 Elsevier Science B.V.

    DOI

  • Assignment of imino proton signals of G-C base pairs and magnesium ion binding: An NMR study of bovine mitochondrial tRNA(GCU)(Ser) lacking the entire D arm

    Hayashi, I, T Yokogawa, G Kawai, T Ueda, K Nishikawa, K Watanabe

    JOURNAL OF BIOCHEMISTRY   121 ( 6 ) 1115 - 1122  1997年06月  [査読有り]

     概要を見る

    The mammalian mitochondrial tRNA(GCU)(Ser) (mt tRNA(GCU)(Ser)) has a unique structure in that it lacks the whole D arm, To elucidate its higher-order structure, we synthesized unmodified bovine mt tRNA(GCU)(Ser) using T7 RNA polymerase and measured its H-1-NMR spectrum in the imino proton region, Although the imino proton signals heavily overlapped, we succeeded in assigning all the seven imino proton signals of the G-C base pairs by a combination of base replacement and N-15-labeling of the G residues of a whole tRNA molecule or of the 3'-half fragment, The results indicate that the tRNA possesses the secondary structure that has been supposed on the basis of biochemical studies, Analysis of the effect of the magnesium concentration on the G-C pairs suggests that the acceptor and T stems do not form a co-axial helix, and that the core region of the tRNA does not interact with magnesium ions, These features are significantly different from those of canonical tRNAs, Despite this, it is very likely that the tRNA as a whole takes a nearly L-shape tertiary structure.

  • Assignment of imino proton signals of G-C base pairs and magnesium ion binding: An NMR study of bovine mitochondrial tRNA(GCU)(Ser) lacking the entire D arm

    Hayashi, I, T Yokogawa, G Kawai, T Ueda, K Nishikawa, K Watanabe

    JOURNAL OF BIOCHEMISTRY   121 ( 6 ) 1115 - 1122  1997年06月  [査読有り]

     概要を見る

    The mammalian mitochondrial tRNA(GCU)(Ser) (mt tRNA(GCU)(Ser)) has a unique structure in that it lacks the whole D arm, To elucidate its higher-order structure, we synthesized unmodified bovine mt tRNA(GCU)(Ser) using T7 RNA polymerase and measured its H-1-NMR spectrum in the imino proton region, Although the imino proton signals heavily overlapped, we succeeded in assigning all the seven imino proton signals of the G-C base pairs by a combination of base replacement and N-15-labeling of the G residues of a whole tRNA molecule or of the 3'-half fragment, The results indicate that the tRNA possesses the secondary structure that has been supposed on the basis of biochemical studies, Analysis of the effect of the magnesium concentration on the G-C pairs suggests that the acceptor and T stems do not form a co-axial helix, and that the core region of the tRNA does not interact with magnesium ions, These features are significantly different from those of canonical tRNAs, Despite this, it is very likely that the tRNA as a whole takes a nearly L-shape tertiary structure.

    DOI

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    19
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    (Scopus)
  • Primary sequence of mitochondrial tRNA(Arg) of a nematode Ascaris suum: Occurrence of unmodified adenosine at the first position of the anticodon

    Y Watanabe, H Tsurui, T Ueda, R FurusihimaShimogawara, S Takamiya, K Kita, K Nishikawa, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1350 ( 2 ) 119 - 122  1997年02月  [査読有り]

     概要を見る

    Mitochondrial tRNA(Arg) from a nematode, Ascaris suum, was purified and sequenced at the RNA level. An unmodified adenosine was found to exist at the anticodon first position, suggesting that, contrary to the conventional wobble rule, the anticodon ACG of the tRNA can translate all the CGN codons.

    DOI DOI2

    Scopus

    23
    被引用数
    (Scopus)
  • Primary sequence of mitochondrial tRNA(Arg) of a nematode Ascaris suum: Occurrence of unmodified adenosine at the first position of the anticodon

    Y Watanabe, H Tsurui, T Ueda, R FurusihimaShimogawara, S Takamiya, K Kita, K Nishikawa, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1350 ( 2 ) 119 - 122  1997年02月  [査読有り]

     概要を見る

    Mitochondrial tRNA(Arg) from a nematode, Ascaris suum, was purified and sequenced at the RNA level. An unmodified adenosine was found to exist at the anticodon first position, suggesting that, contrary to the conventional wobble rule, the anticodon ACG of the tRNA can translate all the CGN codons.

    DOI

  • Mitochondrial methionyl-tRNA transformylase from bovine liver

    Takeuchi, N., Kawakami, M., Ueda, T., Spremulli, L. L., Watanabe, K.

    Nucleic Acids Symp Ser   ( 37 ) 195 - 6  1997年  [査読有り]

  • Essentially minimal sequence for substrate recognition by tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27

    Hori, H., Yamazaki, N., Matsumoto, T., Ueda, T., Nishikawa, K., Kumagai, I., Watanabe, K.

    Nucleic Acids Symp Ser   ( 37 ) 189 - 90  1997年  [査読有り]

  • Colicin E5 as a new type of cytotoxin, which cleaves a specific group of tRNAs.

    Masaki, H., Ogawa, T., Tomita, K., Ueda, T., Watanabe, K., Uozumi, T.

    Nucleic acids symposium series   ( 37 ) 287 - 8  1997年  [査読有り]

  • 5-formylcytidine (f5C) found at the wobble position of the anticodon of squid mitochondrial tRNA(Met)CAU

    Tomita, K., Ueda, T., Watanabe, K.

    Nucleic Acids Symp Ser   ( 37 ) 197 - 8  1997年  [査読有り]

  • Polysemous codon found in Candida species

    Ueda, T., Suzuki, T., Watanabe, K.

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   42 ( 11 ) 1815 - 1827  1997年  [査読有り]

  • Pyridine-promoted factor- and energy-free peptide synthesis systems prepared from various organisms including prokaryote, eukaryote, and mitochondria

    T Nojima, Nitta, I, T Ueda, K Watanabe

    JOURNAL OF BIOCHEMISTRY   119 ( 6 ) 1076 - 1079  1996年06月  [査読有り]

     概要を見る

    We demonstrate here that ribosomes from not only Escherichia coli and Thermus thermophilus [Nitta ef al. (1994) al J. Biochem. 115, 803-807; ibid., (1995) 118, 841-849] but also yeast and bovine mitochondria catalyze peptide synthesis promoted by a high concentration of pyridine in the absence of soluble protein factors and chemical energy sources, and compare some characteristic features of the reactions among these organisms. Sensitivities against antibiotics, chloramphenicol and cycloheximide, showed the same tendency to those in the in vitro aqueous translation systems of these organisms, suggesting that the basic mechanism for peptide synthesis is the same among these organisms. The optimal concentration of pyridine was centered at 50% for all systems, although the dependencies on the pyridine concentrations and the yields of the products were different from one another. All these systems required Mg2+, and only mitochondrial system showed the extra Mn2+-requirement, which enhanced the yield by several fold. The optimum reaction temperatures coincided closely with the growing temperatures of the organisms except for the mitochondrial system, which showed the highest activity above 80 degrees C, The rationale far these observations remains to be solved.

    DOI

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    4
    被引用数
    (Scopus)
  • A new method for identifying the amino acid attached to a particular RNA in the cell

    T Suzuki, T Ueda, K Watanabe

    FEBS LETTERS   381 ( 3 ) 195 - 198  1996年03月  [査読有り]

     概要を見る

    To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3'-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [C-14]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The C-14-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

    DOI DOI2

    Scopus

    17
    被引用数
    (Scopus)
  • A new method for identifying the amino acid attached to a particular RNA in the cell

    T Suzuki, T Ueda, K Watanabe

    FEBS LETTERS   381 ( 3 ) 195 - 198  1996年03月  [査読有り]

     概要を見る

    To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3'-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [C-14]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The C-14-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

    DOI

  • Two nucleotides 5'-adjacent to the anticodon of rat cytoplasmic tRNA(Asp) are not edited

    K Tomita, T Ueda, K Watanabe

    BIOCHIMIE   78 ( 11-12 ) 1001 - 1006  1996年  [査読有り]

     概要を見る

    Cytoplasmic tRNA(Asp) of rat liver was purified by the solid-phase hybridization method and its nucleotide sequence was analyzed by Donis-Keller's method. The results suggested that the two nucleotides next to the anticodon were identical to its gene sequence, a finding that is inconsistent with a previous report demonstrating by several methods that C32 and T33 on the tRNA(Asp) gene are post-transcriptionally converted to U32 and C33, respectively (Beier et al (1992) Nucleic Acids Res 20, 2679-2683). Our results indicate that the tRNA hybridized to an oligonucleotide, designed on the basis of the tRNA(Asp) gene sequence, undergoes no editing and possesses C32 and U33 as predicted from the DNA sequence. Analysis of cDNA synthesized from the purified tRNA(Asp) by the RT-PCR method supported the finding that RNA editing is not involved in the maturation process of rat cytoplasmic tRNA(Asp).

    DOI DOI2

    Scopus

    2
    被引用数
    (Scopus)
  • Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(i)(Met)

    C Ushida, T Muramatsu, H Mizushima, T Ueda, K Watanabe, KO Stetter, PF Crain, JA McCloskey, Y Kuchino

    BIOCHIMIE   78 ( 10 ) 847 - 855  1996年  [査読有り]

     概要を見る

    Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occultum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3' CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal tRNA genes, -50 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a six G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (T-m) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the T-m of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [P-32] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(i)(Met) is due to posttranscriptional modification.

    DOI DOI2

    Scopus

    10
    被引用数
    (Scopus)
  • Two nucleotides 5'-adjacent to the anticodon of rat cytoplasmic tRNA(Asp) are not edited

    K Tomita, T Ueda, K Watanabe

    BIOCHIMIE   78 ( 11-12 ) 1001 - 1006  1996年  [査読有り]

     概要を見る

    Cytoplasmic tRNA(Asp) of rat liver was purified by the solid-phase hybridization method and its nucleotide sequence was analyzed by Donis-Keller's method. The results suggested that the two nucleotides next to the anticodon were identical to its gene sequence, a finding that is inconsistent with a previous report demonstrating by several methods that C32 and T33 on the tRNA(Asp) gene are post-transcriptionally converted to U32 and C33, respectively (Beier et al (1992) Nucleic Acids Res 20, 2679-2683). Our results indicate that the tRNA hybridized to an oligonucleotide, designed on the basis of the tRNA(Asp) gene sequence, undergoes no editing and possesses C32 and U33 as predicted from the DNA sequence. Analysis of cDNA synthesized from the purified tRNA(Asp) by the RT-PCR method supported the finding that RNA editing is not involved in the maturation process of rat cytoplasmic tRNA(Asp).

    DOI

  • Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(i)(Met)

    C Ushida, T Muramatsu, H Mizushima, T Ueda, K Watanabe, KO Stetter, PF Crain, JA McCloskey, Y Kuchino

    BIOCHIMIE   78 ( 10 ) 847 - 855  1996年  [査読有り]

     概要を見る

    Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occultum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3' CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal tRNA genes, -50 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a six G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (T-m) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the T-m of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [P-32] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(i)(Met) is due to posttranscriptional modification.

    DOI

  • Interaction of mitochondrial elongation factors Tu.Ts with aminoacyl-tRNA.

    Benkowski, L.A., Takemoto, C., Ott, G., Beikman, M., Ueda, T., Watanabe, K., Sprinzl, M., Spremulli, L.L.

    Nucleic acids symposium series   ( 33 ) 163 - 6  1995年  [査読有り]

  • Template-dependent Polymerization of Amino Acids by Utilizing Protein Synthesis Systems

    Nitta I, Ueda T, Watanabe K

    Kobunshi   44 ( 9 ) 604 - 607  1995年  [査読有り]

     概要を見る

    配列が制御されたポリペプチドの生産法として,タンパク質合成系の有用性がクローズアップされている。また,芳香族三級アミンを利用したタンパク質合成系の開発を契機に,遺伝情報発現の起源を探ることも夢ではなくなりつつある。本報では,これら応用および基礎の両側面より,タンパク質合成系に関する研究の現状を紹介する。

    DOI CiNii

    Scopus

  • Modified nucleosides of tRNA in decoding

    Ueda, T., Watanabe, K.

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   40 ( 10 ) 1465 - 1473  1995年  [査読有り]

  • A UGU SEQUENCE IN THE ANTICODON LOOP IS A MINIMUM REQUIREMENT FOR RECOGNITION BY ESCHERICHIA-COLI TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE

    S NAKANISHI, T UEDA, H HORI, N YAMAZAKI, N OKADA, K WATANABE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 51 ) 32221 - 32225  1994年12月  [査読有り]

     概要を見る

    Escherichia coli tRNA-guanine transglycosylase is an enzyme which catalyzes replacement of guanine (G(34)) Of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr) by free guanine or free preQ(1) base by a base exchange reaction in the biosynthesis of queuosine (Q) (Okada, N., and Nishimura, S. (1979) J. Biol. Chem. 254, 3061-3066). The gene encoding for this enzyme was amplified from the E. coli genome by polymerase chain reaction and inserted into an overexpression vector, pJLA503, The enzyme was overexpressed by heat induction in E. coli transformed by this recombinant plasmid and purified to homogeneity by two column chromatographies. The sequence requirement in tRNA for recognition by this enzyme was investigated using minihelices corresponding to the anticodon arm of E. coli tRNA(His). Two uridine residues (U-33, U-35) were found to be prerequisite for such recognition by this enzyme, Position 32 required pyrimidines, because the enzyme activity toward the minihelices was markedly reduced or entirely lost when this residue was replaced by purines or was deleted, Adenosine at position 37 and the G(30)-C-40 base pair were not essential despite their conservation. Our results suggest that the enzyme recognizes the U-33-G(34)-U-35 sequence in the anticodon loop and not the tertiary structure of tRNA itself.

  • A UGU SEQUENCE IN THE ANTICODON LOOP IS A MINIMUM REQUIREMENT FOR RECOGNITION BY ESCHERICHIA-COLI TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE

    S NAKANISHI, T UEDA, H HORI, N YAMAZAKI, N OKADA, K WATANABE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 51 ) 32221 - 32225  1994年12月  [査読有り]

     概要を見る

    Escherichia coli tRNA-guanine transglycosylase is an enzyme which catalyzes replacement of guanine (G(34)) Of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr) by free guanine or free preQ(1) base by a base exchange reaction in the biosynthesis of queuosine (Q) (Okada, N., and Nishimura, S. (1979) J. Biol. Chem. 254, 3061-3066). The gene encoding for this enzyme was amplified from the E. coli genome by polymerase chain reaction and inserted into an overexpression vector, pJLA503, The enzyme was overexpressed by heat induction in E. coli transformed by this recombinant plasmid and purified to homogeneity by two column chromatographies. The sequence requirement in tRNA for recognition by this enzyme was investigated using minihelices corresponding to the anticodon arm of E. coli tRNA(His). Two uridine residues (U-33, U-35) were found to be prerequisite for such recognition by this enzyme, Position 32 required pyrimidines, because the enzyme activity toward the minihelices was markedly reduced or entirely lost when this residue was replaced by purines or was deleted, Adenosine at position 37 and the G(30)-C-40 base pair were not essential despite their conservation. Our results suggest that the enzyme recognizes the U-33-G(34)-U-35 sequence in the anticodon loop and not the tertiary structure of tRNA itself.

  • Catalytic role of base nucleotides in peptide bond formation on ribosomes: implication of the origin of protein synthesis

    Takuya Ueda, Itaru Nitta, Takahiko Nojima, Kimitsuna Watanabe

    Nucleic Acids Symposium Series   31   267 - 268  1994年11月

  • Origin and evolution of genetic code: an idea on the mechanism of emergence of unusual genetic codes

    T. Ueda, S. Yokobori, K. Watanabe

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   39 ( 15 ) 2427 - 2437  1994年11月  [査読有り]

    PubMed

  • PRIMARY-ORDER AND HIGHER-ORDER STRUCTURES OF NEMATODE (ASCARIS-SUUM) MITOCHONDRIAL TRANSFER-RNAS LACKING EITHER THE T-STEM OR D-STEM

    YI WATANABE, H TSURUI, T UEDA, R FURUSHIMA, S TAKAMIYA, K KITA, K NISHIKAWA, K WATANABE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 36 ) 22902 - 22906  1994年09月  [査読有り]

     概要を見る

    By fractionation using polyacrylamide gel electrophoresis and/or a preparative hybrid selection method employing solid-phase DNA probes, we prepared and characterized mitochondrial tRNAs from the body wall muscle of Ascaris suum, all of which are thought to lack either the T stem or the D stem from their gene sequences (Okimoto, R., and Wolstenholme, D. R. (1990) EMBO J. 10, 3405-3411). Some of the partially purified tRNAs were appreciably aminoacylated with an extract of A. suum mitochondria. The three species sequenced had CCA sequence at their 3'-ends, and tRNA(Met) had 5-formylcytidine at the anticodon first position, a new modified nucleoside found at the same position of bovine mitochondrial tRNA(Met) (Moriya, J., Yokogawa, T., Wakita, K., Ueda, T., Nishikawa, K., Crain, P. F., Hashizume, T., Pomerantz, S. C., McCloskey, J. A., Kawai, G., Hayashi, N., Yokoyama, S., and Watanabe, K. (1994) Biochemistry 33, 2234-2239). Enzymatic probing of these tRNAs supported the secondary structural model proposed by Okimoto and Wolstenholme in the reference cited above. Chemical probing of tRNA(Phe) demonstrated the existence of tertiary interactions between the (T arm-variable loop)-replacement loop and the D arm. The results suggest that these tertiary interactions enable the bizarre tRNAs of nematode mitochondria to maintain an L-shape-libe structure in order to function in the nematode mitochondrial translation system.

  • PRIMARY-ORDER AND HIGHER-ORDER STRUCTURES OF NEMATODE (ASCARIS-SUUM) MITOCHONDRIAL TRANSFER-RNAS LACKING EITHER THE T-STEM OR D-STEM

    YI WATANABE, H TSURUI, T UEDA, R FURUSHIMA, S TAKAMIYA, K KITA, K NISHIKAWA, K WATANABE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 36 ) 22902 - 22906  1994年09月  [査読有り]

     概要を見る

    By fractionation using polyacrylamide gel electrophoresis and/or a preparative hybrid selection method employing solid-phase DNA probes, we prepared and characterized mitochondrial tRNAs from the body wall muscle of Ascaris suum, all of which are thought to lack either the T stem or the D stem from their gene sequences (Okimoto, R., and Wolstenholme, D. R. (1990) EMBO J. 10, 3405-3411). Some of the partially purified tRNAs were appreciably aminoacylated with an extract of A. suum mitochondria. The three species sequenced had CCA sequence at their 3'-ends, and tRNA(Met) had 5-formylcytidine at the anticodon first position, a new modified nucleoside found at the same position of bovine mitochondrial tRNA(Met) (Moriya, J., Yokogawa, T., Wakita, K., Ueda, T., Nishikawa, K., Crain, P. F., Hashizume, T., Pomerantz, S. C., McCloskey, J. A., Kawai, G., Hayashi, N., Yokoyama, S., and Watanabe, K. (1994) Biochemistry 33, 2234-2239). Enzymatic probing of these tRNAs supported the secondary structural model proposed by Okimoto and Wolstenholme in the reference cited above. Chemical probing of tRNA(Phe) demonstrated the existence of tertiary interactions between the (T arm-variable loop)-replacement loop and the D arm. The results suggest that these tertiary interactions enable the bizarre tRNAs of nematode mitochondria to maintain an L-shape-libe structure in order to function in the nematode mitochondrial translation system.

  • SUBSTRATE-SPECIFICITY OF TRANSFER-RNA (ADENINE-1-)-METHYLTRANSFERASE FROM THERMUS-THERMOPHILUS HB27

    N YAMAZAKI, H HORI, K OZAWA, S NAKANISHI, T UEDA, KUMAGAI, I, K WATANABE, K NISHIKAWA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   58 ( 6 ) 1128 - 1133  1994年06月  [査読有り]

     概要を見る

    tRNA (adenine-1-)-methyltransferase was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB27, by several steps of column chromatographies. The molecular weight of this enzyme was about 60,000 as analyzed by SDS polyacrylamide gel electrophoresis. K-m for E. coli tRNA(2)(Glu) was 100 nM and that for the methyl group donor, S-adenosyl-L-methionine, was 7.8 mu M. The substrate specificity of the enzyme was investigated by using T7 RNA polymerase transcripts and tRNA fragments obtained by partial digestion with RNases. The enzyme was able to transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, however, the extent of methylation was elevated by more than five times when the 5'-half fragment was added and annealed to the 3'-half. This indicates that the main recognition site of the enzyme is within the 3'-half region of tRNA molecule, while the tertiary interaction between the T-loop and the D-loop is very effective for the adequate methylation reaction.

    DOI

  • SUBSTRATE-SPECIFICITY OF TRANSFER-RNA (ADENINE-1-)-METHYLTRANSFERASE FROM THERMUS-THERMOPHILUS HB27

    N YAMAZAKI, H HORI, K OZAWA, S NAKANISHI, T UEDA, KUMAGAI, I, K WATANABE, K NISHIKAWA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   58 ( 6 ) 1128 - 1133  1994年06月  [査読有り]

     概要を見る

    tRNA (adenine-1-)-methyltransferase was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB27, by several steps of column chromatographies. The molecular weight of this enzyme was about 60,000 as analyzed by SDS polyacrylamide gel electrophoresis. K-m for E. coli tRNA(2)(Glu) was 100 nM and that for the methyl group donor, S-adenosyl-L-methionine, was 7.8 mu M. The substrate specificity of the enzyme was investigated by using T7 RNA polymerase transcripts and tRNA fragments obtained by partial digestion with RNases. The enzyme was able to transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, however, the extent of methylation was elevated by more than five times when the 5'-half fragment was added and annealed to the 3'-half. This indicates that the main recognition site of the enzyme is within the 3'-half region of tRNA molecule, while the tertiary interaction between the T-loop and the D-loop is very effective for the adequate methylation reaction.

    DOI

    Scopus

    9
    被引用数
    (Scopus)
  • EFFICIENT EXPRESSION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE GENE BY AN IN-VITRO TRANSLATION SYSTEM USING PHOSPHOROTHIOATE MESSENGER-RNA

    H TOHDA, N CHIKAZUMI, T UEDA, K NISHIKAWA, K WATANABE

    JOURNAL OF BIOTECHNOLOGY   34 ( 1 ) 61 - 69  1994年04月  [査読有り]

     概要を見る

    Dihydrofolate reductase (DHFR) of Escherichia call (E. coli) was synthesized in a cell-free translation system of E. coli directed by phosphorothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of S-P diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates). The molecular weights of the products thus obtained were identical to those with the unsubstituted mRNA. Furthermore, the thio-mRNA for DHFR showed higher translational activities than the corresponding unsubstituted mRNA. It is suggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system. Amongst the various types of thio-mRNAs, the single substitution of adenosine residues was most effective in translational activity. This higher translational activity of thio-mRNA compared with the unsubstituted mRNA was also demonstrated in a continuous flow cell-free system originally developed by Spirin et al. (1988). Therefore, introduction of sulfur atoms into phosphodiester bonds of mRNA appears to be a useful strategy for the stabilization of mRNA in large-scale protein production in vitro.

    DOI DOI2

    Scopus

    11
    被引用数
    (Scopus)
  • EFFICIENT EXPRESSION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE GENE BY AN IN-VITRO TRANSLATION SYSTEM USING PHOSPHOROTHIOATE MESSENGER-RNA

    H TOHDA, N CHIKAZUMI, T UEDA, K NISHIKAWA, K WATANABE

    JOURNAL OF BIOTECHNOLOGY   34 ( 1 ) 61 - 69  1994年04月  [査読有り]

     概要を見る

    Dihydrofolate reductase (DHFR) of Escherichia call (E. coli) was synthesized in a cell-free translation system of E. coli directed by phosphorothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of S-P diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates). The molecular weights of the products thus obtained were identical to those with the unsubstituted mRNA. Furthermore, the thio-mRNA for DHFR showed higher translational activities than the corresponding unsubstituted mRNA. It is suggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system. Amongst the various types of thio-mRNAs, the single substitution of adenosine residues was most effective in translational activity. This higher translational activity of thio-mRNA compared with the unsubstituted mRNA was also demonstrated in a continuous flow cell-free system originally developed by Spirin et al. (1988). Therefore, introduction of sulfur atoms into phosphodiester bonds of mRNA appears to be a useful strategy for the stabilization of mRNA in large-scale protein production in vitro.

    DOI

  • Translational system in animal mitochondria

    Ueda, Takuya, Watanabe, Kimitsuna

    Cell Structure and Function   19 ( 6 ) 428 - 428  1994年  [査読有り]

  • EXISTENCE OF NUCLEAR-ENCODED 5S-RIBOSOMAL-RNA IN BOVINE MITOCHONDRIA

    S YOSHIONARI, T KOIKE, T YOKOGAWA, K NISHIKAWA, T UEDA, K MIURA, K WATANABE

    FEBS LETTERS   338 ( 2 ) 137 - 142  1994年01月  [査読有り]

     概要を見る

    A number of proteins functioning in mitochondria are synthesized in the cytoplasm and imported into the mitochondria via specific transport systems. In mammals, on the contrary, mitochondrial membranes have generally been considered to be impermeable to nucleic acids. However, here we show that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear-encoded 5S RNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5S RNA gene. This RNA molecule was found to be retained in purified bovine mitochondria as well as in the mitoplasts, even after extensive treatment with an RNase, demonstrating that the 5S RNA is actually located inside the mitochondrial inner membrane. The 5S rRNA molecule was also shown to exist in mitochondria from rabbit and chicken.

    DOI DOI2

    Scopus

    59
    被引用数
    (Scopus)
  • EXISTENCE OF NUCLEAR-ENCODED 5S-RIBOSOMAL-RNA IN BOVINE MITOCHONDRIA

    S YOSHIONARI, T KOIKE, T YOKOGAWA, K NISHIKAWA, T UEDA, K MIURA, K WATANABE

    FEBS LETTERS   338 ( 2 ) 137 - 142  1994年01月  [査読有り]

     概要を見る

    A number of proteins functioning in mitochondria are synthesized in the cytoplasm and imported into the mitochondria via specific transport systems. In mammals, on the contrary, mitochondrial membranes have generally been considered to be impermeable to nucleic acids. However, here we show that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear-encoded 5S RNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5S RNA gene. This RNA molecule was found to be retained in purified bovine mitochondria as well as in the mitoplasts, even after extensive treatment with an RNase, demonstrating that the 5S RNA is actually located inside the mitochondrial inner membrane. The 5S rRNA molecule was also shown to exist in mitochondria from rabbit and chicken.

    DOI

  • THE EVOLUTIONARY CHANGE OF THE GENETIC-CODE AS RESTRICTED BY THE ANTICODON AND IDENTITY OF TRANSFER-RNA

    T UEDA, K WATANABE

    ORIGINS OF LIFE AND EVOLUTION OF BIOSPHERES   23 ( 5-6 ) 345 - 364  1993年12月  [査読有り]

     概要を見る

    The discovery of non-universal genetic codes in several mitochondria and nuclear systems during the past ten years has necessitated a reconsideration of the concept that the genetic code is universal and frozen, as was once believed. Here, the flexibility of the relationship between codons and amino acids is discussed on the basis of the distribution of non-universal genetic codes in various organisms insofar as has been observed to date. Judging from the result of recent investigations into tRNA identity, it would appear that the non-participation of the anticodon in recognition by aminoacyl-tRNA synthetase has significantly influenced the variability of codons.

    DOI

  • THE EVOLUTIONARY CHANGE OF THE GENETIC-CODE AS RESTRICTED BY THE ANTICODON AND IDENTITY OF TRANSFER-RNA

    T UEDA, K WATANABE

    ORIGINS OF LIFE AND EVOLUTION OF BIOSPHERES   23 ( 5-6 ) 345 - 364  1993年12月  [査読有り]

     概要を見る

    The discovery of non-universal genetic codes in several mitochondria and nuclear systems during the past ten years has necessitated a reconsideration of the concept that the genetic code is universal and frozen, as was once believed. Here, the flexibility of the relationship between codons and amino acids is discussed on the basis of the distribution of non-universal genetic codes in various organisms insofar as has been observed to date. Judging from the result of recent investigations into tRNA identity, it would appear that the non-participation of the anticodon in recognition by aminoacyl-tRNA synthetase has significantly influenced the variability of codons.

    DOI

    Scopus

    7
    被引用数
    (Scopus)
  • EFFECTS OF POLYAMINES ON A CONTINUOUS CELL-FREE PROTEIN-SYNTHESIS SYSTEM OF AN EXTREME THERMOPHILE, THERMUS-THERMOPHILUS

    T UZAWA, A YAMAGISHI, T UEDA, N CHIKAZUMI, K WATANABE, T OSHIMA

    JOURNAL OF BIOCHEMISTRY   114 ( 5 ) 732 - 734  1993年11月  [査読有り]

     概要を見る

    A continuous cell-free protein synthesis system of an extremely thermophilic eubacterium, Thermus thermophilus HB27, was constructed. This system produced MS2 phage RNA translation products at a rate of more than 5 mu g per hour per 1.9 mg of ribosomes at 65 degrees C and the production continued linearly for at least 340 min. When no polyamine was added, the system did not produce the proteins. The highest activity was recorded when O.1 mM tetrakis(3-aminopropyl)ammonium and 1.0 mM spermine were added simultaneously.

    DOI

    Scopus

    16
    被引用数
    (Scopus)
  • MOLECULAR MECHANISM OF THE GENETIC-CODE VARIATIONS FOUND IN CANDIDA SPECIES AND ITS IMPLICATIONS IN EVOLUTION OF THE GENETIC-CODE

    K WATANABE, T UEDA, T YOKOGAWA, T SUZUKI, K NISHIKAWA, M MORI, T OHAMA, H NAKABAYASHI, T NAKASE, S OSAWA

    TRANSLATIONAL APPARATUS     647 - 656  1993年  [査読有り]

  • Tertiary structures of mitochondrial tRNAs having characteristic secondary structures

    Watanabe, Y., Kawai, G., Yokogawa, T., Hayashi, I., Hayashi, N., Nishikawa, K., Ueda, T., Watanabe, K.

    Nucleic Acids Symp Ser   ( 29 ) 209 - 10  1993年  [査読有り]

  • THE GENE FOR SERINE TRANSFER-RNA HAVING ANTICODON SEQUENCE CAG IN A PATHOGENIC YEAST, CANDIDA-ALBICANS

    T SUZUKI, T UEDA, T OHAMA, S OSAWA, K WATANABE

    NUCLEIC ACIDS RESEARCH   21 ( 2 ) 356 - 356  1993年01月  [査読有り]

    DOI

    Scopus

    8
    被引用数
    (Scopus)
  • CODONS AGA AND AGG ARE READ AS GLYCINE IN ASCIDIAN MITOCHONDRIA

    S YOKOBORI, T UEDA, K WATANABE

    JOURNAL OF MOLECULAR EVOLUTION   36 ( 1 ) 1 - 8  1993年01月  [査読有り]

     概要を見る

    A 1.2-kb DNA fragment of the cytochrome oxidase subunit I (CO I) gene of mitochondria isolated from an ascidian, Halocynthia roretzi, was amplified by polymerase chain reaction (PCR) and sequenced. Codons AGA and AGG appeared in its reading frame, indicating that these are sense codons in this organelle. Sequence comparisons with the corresponding regions of other animal mitochondrial CO I genes suggest that codons AGA and AGG correspond to glycine in the ascidian mitochondrial genome, but not to serine as in most invertebrate genomes, nor to stops as in vertebrate genomes. The other codons are identical to those of vertebrate mitochondria.

    DOI DOI2

    Scopus

    42
    被引用数
    (Scopus)
  • Tertiary structural analysis of Escherichia coli lysine tRNA.

    Hayashi, N., Kawai, G., Takayanagi, M., Noguchi, T., Ueda, T., Nishikawa, K., Miura, K., Miyazawa, T., Yokoyama, S., Watanabe, K.

    Nucleic acids symposium series   ( 29 ) 195 - 196  1993年  [査読有り]

  • Non-universal genetic codes and their evolutionary processes

    Ueda, T., Watanabe, K.

    Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme   38 ( 16 ) 2677 - 2691  1993年  [査読有り]

  • CODONS AGA AND AGG ARE READ AS GLYCINE IN ASCIDIAN MITOCHONDRIA

    S YOKOBORI, T UEDA, K WATANABE

    JOURNAL OF MOLECULAR EVOLUTION   36 ( 1 ) 1 - 8  1993年01月  [査読有り]

     概要を見る

    A 1.2-kb DNA fragment of the cytochrome oxidase subunit I (CO I) gene of mitochondria isolated from an ascidian, Halocynthia roretzi, was amplified by polymerase chain reaction (PCR) and sequenced. Codons AGA and AGG appeared in its reading frame, indicating that these are sense codons in this organelle. Sequence comparisons with the corresponding regions of other animal mitochondrial CO I genes suggest that codons AGA and AGG correspond to glycine in the ascidian mitochondrial genome, but not to serine as in most invertebrate genomes, nor to stops as in vertebrate genomes. The other codons are identical to those of vertebrate mitochondria.

    DOI

  • Purification and characterization of tRNA(adenosine-1-)-methyltransferase from Thermus thermophilus HB27

    Yamazaki, N., Hori, H., Ozawa, K., Nakanishi, S., Ueda, T., Kumagai, I., Watanabe, K., Nishikawa, K.

    Nucleic Acids Symp Ser   ( 27 ) 141 - 2  1992年  [査読有り]

  • Structure of mitochondrial tRNA

    Yokogawa, T., Watanabe, Y., Yotsumoto, Y., Kumazawa, Y., Ueda, T., Hirao, I., Miura, K., Watanabe, K.

    Nucleic Acids Symp Ser   ( 25 ) 175 - 6  1991年  [査読有り]

  • Cell-free translation system using phosphorothioate-containing mRNA

    Ueda, T., Tohda, H., Chikazumi, N., Eckstein, F., Watanabe, K.

    Nucleic Acids Symp Ser   ( 25 ) 151 - 2  1991年  [査読有り]

  • THE GENETIC-CODE OF A SQUID MITOCHONDRIAL GENE

    T SHIMAYAMA, H HIMENO, J SASUGA, T UEDA, K WATANABE

    SYMPOSIUM ON NUCLEIC ACIDS TECHNOLOGY   22 ( 22 ) 73 - 74  1990年  [査読有り]

  • DISCRIMINATOR BASE AND ANTICODON AS A TRANSFER-RNA IDENTITY ELEMENT

    H HIMENO, H ASAHARA, K TAMURA, T HASEGAWA, T UEDA, K WATANABE, M SHIMIZU

    SYMPOSIUM ON NUCLEIC ACIDS TECHNOLOGY   22 ( 22 ) 117 - 118  1990年  [査読有り]

  • SOME PROPERTIES OF BOVINE MITOCHONDRIAL SERINE TRANSFER-RNA GENE TRANSCRIPT SYNTHESIZED WITH T7 RNA-POLYMERASE

    T UEDA, K WATANABE

    SIXTEENTH SYMPOSIUM ON NUCLEIC ACIDS CHEMISTRY   21 ( 21 ) 113 - 114  1989年  [査読有り]

  • A novel lysine-substituted nucleoside in the first position of the anticodon of minor isoleucine tRNA from Escherichia coli

    T. Muramatsu, S. Yokoyama, N. Horie, A. Matsuda, T. Ueda, Z. Yamaizumi, Y. Kuchino, S. Nishimura, T. Miyazawa

    Journal of Biological Chemistry   263 ( 19 ) 9261 - 9267  1988年  [査読有り]

     概要を見る

    A minor species of isoleucine tRNA (tRNA(minor)(Ile)) specific to the codon AUA has been isolated from Escherichia coli B and a modified nucleoside N+ has been found in the first position of the anticodon (Harada, F., and Nishimura, S. (1974) Biochemistry 13, 300-307). In the present study, tRNA(minor)(Ile) was purified from E. coli A19, and nucleoside N+ was prepared, by high-performance liquid chromatography, in an amount (0.6 A260 units) sufficient for the determination of chemical structures. By 400 MHz 1H NMR analysis, nucleoside N+ was found to have a pyrimidine moiety and a lysine moiety, the ε-amino group of which was involved in the linkage between these two moieties. From the NMR analysis together with mass spectrometry, the structure of nucleoside N+ was determined as 4-amino-2-(N6-lysino)-1-(β-D-ribofuranoysl)pyrimidinium ('lysidine'), which was confirmed by chemical synthesis. Lysidine is a novel type of modified cytidine with a lysine moiety and has one positive charge. Probably because of such a unique structure, lysidine in the first position of anticodon recognizes adenosine but not guanosine in the third position of codon.

    PubMed

  • LARGE-SCALE ISOLATION AND SOME PROPERTIES OF AGY-SPECIFIC SERINE TRANSFER-RNA FROM BOVINE HEART-MITOCHONDRIA

    T UEDA, T OHTA, K WATANABE

    JOURNAL OF BIOCHEMISTRY   98 ( 5 ) 1275 - 1284  1985年  [査読有り]

  • Structural analysis of bovine mitochondrial tRNASer (AGY)

    Ueda, T., Watanabe, K., Ohta, T.

    Nucleic Acids Symp Ser   ( 12 ) 141 - 4  1983年  [査読有り]

  • Primary sequence of ascidian mitochondrial glycine tRNA translating non-universal codons AGR (R: A, G)

    Akiko Kondo, Shin-ichi Yokobori, Takuya Ueda, Kimitsuna Watanabe

    Nucleic Acids Symp. Ser.   35   279 - 280  [査読有り]

▼全件表示

書籍等出版物

  • Molecular mechanism of the genetic code variations found in Candida species and its implications in evolution of the genetic code

    The translational apparatns Structure, Function, Regulation, Evolution  1993年

共同研究・競争的資金等の研究課題

  • 人工細胞を指向したバイオシステムの無細胞合成系の開発

    日本学術振興会  科学研究費助成事業

    研究期間:

    2016年04月
    -
    2019年03月
     

    上田 卓也

     概要を見る

    人工細胞を目標として、再構築型遺伝子発現系PURE systemによるバイオシステムの構築を進めた。細胞分裂システムを、リポソーム内で合成し、変形するリポソームを作製した。ATP合成酵素とバクテリオロドプシンを、リポソーム内で発現させ、光依存的なエネルギー生産システムを構築した。リボソームの小サブユニットについては、生合成因子の存在下、生理的条件下でのアセンブリー系を構築した。大サブユニットについては個別精製したリボソームタンパク質とrRNAから、タンパク質合成活性を有するサブユニットの再構成に成功した。また、転写されたtRNA分子よるPURE systemを開発した。

  • 冥王代プレ生命システムの実験室内再現

    日本学術振興会  科学研究費助成事業

    研究期間:

    2015年04月
    -
    2017年03月
     

    上田 卓也

     概要を見る

    人工の脂質膜であるNanodiscを用いたRibosome Displayの確立に取り組んだ。ribosomeから提示されたペプチド鎖が、脂質との親和性によって選抜されることを示すために、7回膜貫通型タンパク質bacteriorhodopsin(bR)と、FtsZ interacting protein A(ZipA)の一回膜貫通ドメインを膜タンパク質のモデル分子として使用した。可溶性のタンパク質(DHFR)と、各モデル分子を系中に共存させたときに、サイクル数の増加に伴ってモデル分子の遺伝子が濃縮されることが示された。このことにより膜タンパク質のibosome Displayによる選抜が可能であり、また脂質としてNanodiscを用いることが有効であることが示されたため、この手法を用いて、ランダムな10アミノ酸のペプチドをコードするDNAライブラリーを構築した後、PURE systemで翻訳し、Nanodiscとの親和性を有する翻訳複合体の選抜を行った。1サイクル後のmRNAの配列解析の結果、コドンの出現確率に対するアミノ酸の出現頻度において、イソロイシンの相対頻度が顕著に上昇し、グリシンの相対頻度が顕著に減少した。このサイクルを繰り返すことによって、原始膜タンパク質の配列に迫ることができるものと期待できる。
    大腸菌の細胞分裂の根幹を担う3つのタンパク質を、人工膜小胞中でPURE systemを用いてde novoに合成することに成功した。FtsZは、脂質膜にアンカリングされたZipAと結合、重合し、脂質膜の変形を惹起した。このリポソームは分裂する人工細胞の開発につながるものと期待できる。

  • 合成生物学的な手法による人工光合成システムの開発

    日本学術振興会  科学研究費助成事業

    研究期間:

    2014年04月
    -
    2016年03月
     

    上田 卓也

     概要を見る

    バクテリオロドプシンとFoF1-ATP合成酵素を組み込んだ(BR-FoF1)リポソームベースのバイオナノリアクターの構築が研究目的である。PURE systemによりFoF1-ATP合成酵素の全サブユニットをリポソーム上に発現させた。特に、各サブユニットの合成量が等しくなるように、DNA量の条件検討を行った。合成されたATP合成酵素が十分な活性を有していることを見いだした。バクテリオロドプシンについては、レチナールのスペクトルからナノディスク上には活性を有した形で挿入されていることが示された。さらに、変異型と天然型のバクテリオロドプシンをリポソームディスプレイ法による選抜が可能であった。

  • 合成生物学的手法による超分子複合体形成の研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    2011年04月
    -
    2014年03月
     

    上田 卓也

     概要を見る

    多くの蛋白質は複合体を形成することで制御された機能発現が可能となるが、細胞内の複合体の形成プロセスはほとんど未解明である。本プロジェクトでは、再構築型無細胞蛋白質合成系PURE systemを用いて、ポリペプチドの合成反応と複合体形成を試験管内で再構築を行った。その結果、細胞膜上の22のポリペプチドから形成されるATP合成酵素、および3つのポリペプチドからなるトランスロコンの合成に成功した。また16SrRNAと20種類の蛋白質からなる超分子複合体であるリボソームの小サブユニット(30S)の効率の高い再構築を、さまざまな生合成因子の共存により成功した。

  • 翻訳と共役した無細胞蛋白質成熟システムの構築

    日本学術振興会  科学研究費助成事業

    研究期間:

    2006年
    -
    2008年
     

    上田 卓也, 清水 義宏

     概要を見る

    蛋白質の成熟過程のメカニズム解明することを目標として、再構築した蛋白質合成系PURE systemによって大腸菌ゲノム上の全蛋白質(4132個)の合成を行い、各蛋白質の凝集特性を遠心分離により可溶性を評価した。PURE systemにより合成可能また電気泳動可能であった7割についての評価したところ、凝集しやすさは二峰性を示すことが示された。分子量が小さい蛋白質、等電点が低い蛋白質は可溶性が高いことが示された。また、細胞質の凝集傾向のある蛋白質792個について、シャペロン存在下のPURE systemで合成を行い、その可溶性の向上から、シャペロン依存性を評価した。以上の解析結果をe-Solデータベースとして公開した(http://tp-esol.genes.nig.ac.jp/)。

  • セルフリーイムノロジーによる人工抗体作製法の開発

    日本学術振興会  科学研究費助成事業

    研究期間:

    2006年
     
     
     

    上田 卓也

     概要を見る

    リボソームディスプレー法(RD法)を行う場合、リボソーム三者複合体の形成効率が極めて重要なポイントとなる。そこでまず、形成効率に影響するC末端側コンストラクトの検討を行った。HyHEL10scFv(リゾチーム特異的抗体)の下流に、リボソーム内部で翻訳アレストを起こすと報告されているSecMのアレスト配列を導入することで、RDにおけるmRNAの効率が上昇するという新しい知見を得るに至った。これはSecM導入によるリボソーム複合体の形成効率向上によるものであると考えられ、特にPURESYSTEMではSecMのストールを不安定化するtmRNAなどが存在しないため有効な方法であると推測された。また、PURESYSTEMは因子の有無など環境を自由に設計できるため、これまでの細胞抽出系を用いた場合では不可能であったシステムの詳細な素過程解析を行うことが可能である。「mRNA-リボソーム-蛋白質複合体」の形成に必須な各因子(EF、リボソーム、mRNA)をRD系中から出し入れし、HyHEL10とDHFRとの1:30からのセレクション(リガンドとしてリゾチームを使用)を行ったところ全ての因子が存在する場合にのみ、HyHEL10の特異的選択がみられ、「mRNA-リボソーム-蛋白質複合体による目的分子の特異的選択」というRD法の基本概念を、PURESYSTEMを用いることで実験的に確認することが出来た。さらに、scFvの特異的選択系において、細胞抽出液の無細胞蛋白質合成系を用いた場合よりもPURESYSTEMを用いた場合の方が、高効率に選択されることを実験的に確認した。また、本方法では1ラウンドのセレクションにおいて、目的分子を12000倍以上に濃縮できることを確認した。これまで報告されているRD法では1000倍以下の濃縮効率である。さらに、プールとして、10^<10>から10^<11>倍の非結合性のタンパク質遺伝芋で希釈しても、目的の抗体遺伝子が、3回から5回の選抜で単離可能なことがしめされ、これは本方法の有効性を示すものであると考えられる。さらに、抗原の固定について、PURE systemを用いて非天然アミノ酸を導入して効率よく固定する手法を開発した。

  • 試験管内遺伝子発現系を用いたタンパク質の機能発現プロセスの研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    2005年
    -
    2006年
     

    上田 卓也

     概要を見る

    リボソーム上で合成された新生ペプチドは、複雑なプロセスをへて、機能を持つ多様な形態の成熟型蛋白質となる。私たちは、近年、翻訳過程に必須な因子のみ構成された試験管内遺伝子発現系PUREsystemを再構築し、蛋白質の誕生プロセスを無細胞化した。このシステムの発展型として蛋白質成熟プロセスに関与する因子群をPURESYSTEMに共存させた複合型無細胞システムを開発し、蛋白質の成熟過程のメカニズム解明することを本研究の目標としている。
    まず、新生蛋白質のフォールディングにおける分子シャペロンの役割、および翻訳と共役したフォールディングプロセスを解析するために、フォールディングに関与するシャペロンの共存させたPURESYSTEMを構築した。従来のシャペロンのフォールディングへの関与に関する仮説を検証することを目的として、このシステムを用いた解析を進めている。単鎖抗体(scFv)を用いた実験では、DnaKシステム(DnaK-J/GrpE)とトリガーファクターは、新生蛋白質の可溶化率とその抗原結合活性の向上に著しく効果があり、合成されたscFvの正確な折り畳みを促進することが示された。また、トリガーファクターとDnaKシステムがco-translationalおよびpost-translationalの両プロセスで新生蛋白質の凝集を抑えることが明らかとなった。また、これらのシャペロンの基質となる蛋白質を、大腸菌遺伝子から検索し、シャペロンニン(GroEL/ES)に依存してフォールディングする基質蛋白質-stringent substrate-を見いだした。GroEL/ES-dependentフォールディングプロセスをin vitroで解析したところ、今まで言われたpost-translationalプロセスではなく、新生ポリペプチドーリボソーム-mRNA複合体に結合し、co-translationalプロセスでフォールディングに寄与する可能性が高いことが示された。またsingle-ringのGroELを用いた実験から、GroELが翻訳途中の新生ペプチドに結合し、翻訳終了後にGroESがGroELに結合し、フォールディングを誘導することが示された。

  • 翻訳における終結と開始の連携機構の解明

    日本学術振興会  科学研究費助成事業

    研究期間:

    2005年
    -
    2006年
     

    上田 卓也

     概要を見る

    私達は、大腸菌の翻訳因子やリボソームを完全に精製し、再構築したPURE(Protein synthesizing system Using Recombinant Elements)システムを完成させることに成功している。PUREシステムによって翻訳のすべてのプロセスを、試験管のなかで行うことが可能であるばかりではなく、すべてのプロセスのスナッブショットを取ることが可能である。本研究では、RF1とRF2による終止コドンのデコーディングの機構をPUREシステムにより明らかにすることを目標としている。PUREシステムを用いた実験系によって、RF1とRF2の、ペブチドアンチコドン部位に変異を導入し、これらのRFの終止コドンの解読の特性を解析した。その結果、これらのペプチドアンチコドンの領域は、終止コドンの解読特性には関与しないことが示唆された。また、RRFは、翻訳終結後のリボソーム・訳NA複合体を解消のプロセスの解析を行った。その結果、RRFは、リボソームを樹来考えられていた30S、50Sの解離した形ではなく、70Sの状態で解離させることが示された。また、この終結プロセスにおいて、サブユニットの会合の安定性にスペルミジンなどのポリアミンが重要であることが示唆された。さらに、PURE systemを用いて、trans-translationのメカニズム解明を行い、smpBがtmRNAのコドンアンチコドンの相互作用を機能的に補う働きがあることが示された。

  • 疾患関連蛋白質の生産技術の開発

    日本学術振興会  科学研究費助成事業

    研究期間:

    2004年
     
     
     

    上田 卓也, 田口 英樹, 富田 野乃

     概要を見る

    蛋白質を合成する場合に遭遇する困難は、そのフォールディングの問題である。PUREシステムに、さまざまの分子シャペロンのサブシステムを共存させ、フォールディングの制御を行う。時に、凝集の抑制することはきわめて重要である。すでに、蛋白質の新生ペプチドに結合することが知られているDnaK関連のシャペロンの共存システム構築に成功した。また、GroELは翻訳とカップルして蛋白質のフォールディングに関与に関与することを見いだした。また、大腸菌ゲノムにコードされる蛋白質遺伝子について、GroELとDnaKの基質の網羅的検索を行い、stringent substrateの同定に成功した。ゲノム上の蛋白質の3割は、膜蛋白質もしくは分泌蛋白質であるとされている。また、情報伝達において膜上のリセプターからその情報伝達が開始される。従って、膜蛋白質や分泌蛋白質などの細胞質以外で機能する蛋白質の解析は疾患の病理解明において重要であり、また薬剤の開発にはこうした蛋白質の機能と構造の研究が不可欠である。PUREシステムに反転膜小胞を連列したシステムの構築を行い、膜蛋白質を高効率で合成可能な系を開発することに成功した。

  • 可変領域の安定性を利用した抗原濃度測定法の実用化

    日本学術振興会  科学研究費助成事業

    研究期間:

    2002年
    -
    2004年
     

    上田 宏, 長棟 輝行, 上田 卓也

     概要を見る

    1.繊維状ファージを用いた新規な蛋白質間相互作用測定系split Fv (spFv)システムの確立
    ファージに二種類の蛋白質の片方のみを提示し,片方を分泌型蛋白質として産生させ両者の間の相互作用を測定することができる新規蛋白質間相互作用測定系split Fv (spFv)システムを構築した.これを各種の抗体可変領域VH/VL間の相互作用測定に応用したところ,モデル系で抗原添加に伴う相互作用変化を測定できた(オープンサンドイッチ法,OS法).さらにこのシステムでは二つのドメインVH, VL両者を同時に提示して抗原でパニングすることも可能であり,実際にフレームワーク領域のライブラリを作製し,これまで不明であった抗原結合能とVH/VL相互作用の強弱との関係,およびVH/VL間の相互作用を決定づける残基を明らかにした。
    2.低分子の非競合的免疫測定法としてのOS法の確立
    上記のSpFvシステムを用いて,各種低分子(ビスフェノールA,11-デオキシコルチゾール,ホスホチロシン,ジベレリンA24,オステオカルシンペプチド等)に対する抗体遺伝子クローニングと,OS法による非競争的免疫測定に成功した。いずれの抗原検出においても従来の競合法に比べ,高い検出感度と広い測定濃度域を得ることができた。また得られた遺伝子を用いてVHとアルカリフォスファターゼとの融合蛋白質,およびMBP-VL融合蛋白質等の精製蛋白質を作製し,これらを用いて実用的な測定系を組むことができた。
    3.真核細胞を用いた抗原特異的抗体発現細胞選択系の開発
    抗原を認識して活性化し,増殖信号を伝達できる細胞表面キメラ受容体を発現させたIL-3依存性細胞株を用いて,抗原特異的な抗体断片を選択し,かつ細胞にCre recombinaseを発現させることで培養上清に糖鎖修飾scFvを分泌できるシステム(ASAPシステム)を構築した。

  • 新規リボソームディスプレー法によるプロテインネットワークの研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    2002年
    -
    2004年
     

    上田 卓也, 上田 宏, 富田 野乃

     概要を見る

    リボソームディスプレー法(RD法)を行う場合、リボソーム三者複合体の形成効率が極めて重要なポイントとなる。そこでまず、形成効率に影響するC末端側コンストラクトの検討を行った。HyHEL10scFv(リソチーム特異的抗体)の下流のスペーサー配列Gene[]のC末端側に、リボソーム内部で翻訳アレストを起こすと報告されているSecMのアレスト配列FXXXXWIXXXXGIRAGPを導入することで、RDにおけるmRNAの効率が上昇するという新しい知見を得るに至った。これはSecM導入によるリボソーム複合体の形成効率向上によるものであると考えられ、特にPURESYSTEMではSecMのストールを不安定化するtmRNAなどが存在しないため有効な方法であると推測された。また、PURESYSTEMは因子の有無など環境を自由に設計できるため、これまでの細胞抽出系を用いた場合では不可能であったシステムの詳細な素過程解析を行うことが可能である。「mRNA-リボソームー蛋白質複合体」の形成に必須な各因子(EF、リボソーム、mRNA)をRD系中から出し入れし、HyHEL10とDHFRとの1:30からのセレクション(リガンドとしてリソチームを使用)を行ったところ全ての因子が存在する場合にのみ、HyHEL10の特異的選択がみられ、「mRNA-リボソームー蛋白質複合体による目的分子の特異的選択」というRD法の基本概念を、PURESYSTEMを用いることで実験的に確認することが出来た。さらに、scFvの特異的選択系において、細胞抽出液の無細胞蛋白質合成系を用いた場合よりもPURESYSTEMを用いた場合の方が、高効率に選択されることを実験的に確認した。また、本方法では1ラウンドのセレクションにおいて、目的分子を12000倍以上に濃縮できることを確認してる。これまで報告されているRD法では1000倍以下の濃縮効率であるので、これは本方法の有効性を示すものであると考えられる。

  • 試験管内遺伝子発現系を用いたタンパク質の機能発現プロセスの研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    2003年
     
     
     

    上田 卓也, 富田 野乃

     概要を見る

    特定領域研究(2)(試験管内遺伝子発現系を用いたタンパク質の機能発現プロセスの研究)
    私達は、大腸菌の翻訳因子やリボソームを完全に精製し、再構築したPURE(Protein synthesizing system Using Recombinant Elements)システムを完成させることに成功している。PUREシステムによって翻訳のすべてのプロセスを、試験管のなかで行うことが可能であるばかりではなく、すべてのプロセスのスナップショットを取ることが可能である。本研究では、PUREシステムを用いて、翻訳過程と蛋白質のフォールディングのプロセスがいかに共同的に働いているかを検討した。大腸菌において、新生ペプチドがリボソーム上で合成される場合、トリッガーファクター、DnaK、DnaJ、GrpE、GroELが関与してフォールディングが進行するとする説が提唱されている。
    まず、PUREシステムで合成したscFv蛋白質は、通常の抽出液を用いたものに比べ,凝集が低く押さえられることを見いだした。私達は、抗体の一部であるscFvを、PUREシステムを用いてこれらのシャペロン存在下で蛋白質の合成を行った。その結果、DnaK,GrpE存在下において凝集する蛋白質の比率が低下し、またフォールングが促進されることが、確認された。また、GroEL/ESが、翻訳と共同している可能性も示唆された。また、Apg2などの真核生物由来のシャペロンも高効率で凝集を防ぐことが示された。また、ディスルフィド結合を促進するPDIが酸化的な環境でscFVの活性を向上させることを見いだした。同時に膜蛋白質の合成系も確立し、SecBによる凝集の防止が、分泌には必須であるごとが明らかとなった。

  • 翻訳の終結機構の解明

    日本学術振興会  科学研究費助成事業

    研究期間:

    2003年
     
     
     

    上田 卓也

     概要を見る

    特定領域(2)(翻訳の終結機構の解明)
    翻訳のメカニズムは解明されたとされている。しかし、分子メカニズムに関しては実に不明な点が多く見いだされる。特に、翻訳終結から開始へのプロセスに関しては、むしろ推定の域を出ないものが多い。私達は、大腸菌の翻訳因子やリボソームを完全に精製し、再構築したPURE(Protein synthesizing system Using Recombinant Elemehts)システムを完成させることに成功している。PUREシステムによって翻訳のすべてのプロセスを、試験管のなかで行うことが可能であるばかりではなく、すべてのプロセスのスナッブショットを取ることが可能である。本研究では、RF1とRF2による終止コドンのデコーディングの機構をPUREシステムにより明らかにすることを目標としている。14年度は、PUREシステムを用いた実験系によって、RF1とRF2の、ペブチドアンチコドン部位に変異を導入し、これらのRFの終止コドンの解読の特性を解析した。その結果、これらのペプチドアンチコドンの領域は、終止コドンの解読特性には関与しないことが示唆された。
    同時にRRFは、翻訳終結後のリボソーム・mRNA複合体を解消のプロセスの解析を行った。その結果RRFは、リボソームを従来考えられていた30S、50Sの解離した形ではなく、70Sの状態で解離させることが示された。また、70Sの各サブユニットへの解離はIF3によっておこることが示された。

  • 試験管内遺伝子発現系を用いたタンパク質の機能発現プロセスの研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    2002年
     
     
     

    上田 卓也, 富田 野乃

     概要を見る

    私達は、大腸菌の翻訳因子やリボソームを完全に精製し、再構築したPURE(Protein synthesizing system Using Recombinant Elements)システムを完成させることに成功している。PUREシステムによって翻訳のすべてのプロセスを、試験管のなかで行うことが可能であるばかりではなく、すべてのプロセスのスナッブショットを取ることが可能である。本研究では、PUREシステムを用いて、翻訳過程と蛋白質のフォールディングのプロセスがいかに共同的に働いているかを検討した。大腸菌において、新生ペプチドがリボソーム上で合成される場合、トリッガーファクター、DnaK、DnaJ、GrpE、GroELが関与してフォールディングが進行するとする説が提唱されている。まず、PUREシステムで合成したscFv蛋白質は、通常の抽出液を用いたものに比べ、凝集が低く押さえられることを見いだした。私達は、抗体の一部であるscFvを、PUREシステムを用いてこれらのシャペロン存在下で蛋白質の合成を行った。その結果、DnaK, GrpE存在下において凝集する蛋白質の比率が低下し、またフォールングが促進されることが、確認された。また、GroEL/ESが、翻訳と共同している可能性も示唆された。また、Apg2などの真核生物由来のシャペロンも高効率で凝集を防ぐことが示された。また、ディスルフィド結合を促進するPDIが酸化的な環境でscFVの活性を向上させることを見いだした。同時に膜蛋白質の合成系も確立し、SecBによる凝集の防止が、分泌には必須であることが明らかとなった。

  • 翻訳の終結機構の解明

    日本学術振興会  科学研究費助成事業

    研究期間:

    2002年
     
     
     

    上田 卓也

     概要を見る

    翻訳のメカニズムは解明されたとされている。しかし、分子メカニズムに関しては実に不明な点が多く見いだされる。特に、翻訳終結から開始へのプロセスに関しては、むしろ推定の域を出ないものが多い。私達は、大腸菌の翻訳因子やリボソームを完全に精製し、再構築したPURE(Protein synthesizing system Using Recombinant Elements)システムを完成させることに成功している。PUREシステムによって翻訳のすべてのプロセスを、試験管のなかで行うことが可能であるばかりではなく、すべてのプロセスのスナッブショットを取ることが可能である。本研究では、RF1とRF2による終止コドンのデコーディングの機構をPUREシステムにより明らかにすることを目標としている。14年度は、PUREシステムを用いた実験系によって、RF1とRF2の、ペブチドアンチコドン部位に変異を導入し、これらのRFの終止コドンの解読の特性を解析した。その結果、これらのペプチドアンチコドンの領域は、終止コドンの解読特性には関与しないことが示唆された。
    同時にRRFは、翻訳終結後のリボソーム・mRNA複合体を解消のプロセスの解析を行った。その結果、RRFは、リボソームを樹来考えられていた30S、50Sの解離した形ではなく、70Sの状態で解離させることが示された。また、70Sの各サブユニットへの解離はIF3によっておこることが示された。

  • Whole-in-oneシステムによる生命の創成

    日本学術振興会  科学研究費助成事業

    研究期間:

    2000年
     
     
     

    上田 卓也

     概要を見る

    大腸菌の蛋白質合成系に関与する可溶性の蛋白性因子(開始因子、伸長因子、終結因子、アミノアシル合成酵素等)の31種類の因子をクローニング、大量発現、精製を行った。さらに、リボソームも極めて純度のものを単離し、また80種類のtRNAを含む画分を調整した。これらの170種類の分子種から再構築することにより、蛋白質合成活性をもった蛋白質合成系(PUREシステム)の作製に成功した。PUREシステムは、1mlあたり0.2mgの蛋白質の生産が可能であった。現在生体外蛋白質合成系または無細胞蛋白質合成系と称されているものは、すべて細胞を破砕した抽出液に過ぎず、真の意味での生体外蛋白質合成系は申請者の本システムがはじめてである。このシステムは、蛋白質分解酵素や核酸分解酵素を含まず、また実験者がその反応を自由に制御できる点で、大変優れた遺伝子発現システムの構築に成功したといえる。このシステムにより、MS2やQβなどのファージを創製することを試みたが、感染能を有するファージを造り出すことはできなかった。同時の並行して、大腸菌の抽出液を用いたシステムでは、感染能を有するファージが生産された。このことから、こうしたファージのアセンブリーには大腸菌由来の蛋白質が関与していることが、示唆された。

  • RNAをターゲティングする蛋白質の検索システムのデザイン

    日本学術振興会  科学研究費助成事業

    研究期間:

    2000年
     
     
     

    上田 卓也

     概要を見る

    遺伝情報発現の転写レベルでの制御の全容が、真核生物を中心として明らかになりつつあるが、翻訳レベルでの制御機構に関しては、その解明はいまだ十分とは言い難い。翻訳段階での制御は、転写とは異なり、短時間かつデリケートに行えるために、利点が多いと考えられ,今後多様な制御機構が明らかにされるものと予想される。本研究では、RNAに結合能を持つ蛋白質を、スクリーニングするシステムを構築することにより、翻訳レベルでの制御機構の全体像に迫ることに挑戦する。すでに大腸菌の精製した必須な成分から再構築した無細胞翻訳系(PURE SYSTEM)の開発に成功している。このPURE SYSTEMには、蛋白質やRNAなどを分解する成分が含まれないことから、今後ポリソームディスプレー法の主流となるものと期待されている。本研究ではPUREシステムを翻訳制御の研究における中心的なスクリーニングの系として確立することを目的とする。まず,PURE SYSTEMにより、cDNAのラブラリーから、蛋白質を合成させ、新生ペプチドの中から、mRNAに結合能を持つものを選抜する。あらかじめcDNAには、タグ配列を挿入しておき、このタグを利用して、ペプチドと結合したmRNAを選抜し、RT-PCR、PCRにより増幅する。このサイクルを繰り返すことで、自らのmRNAに結合する蛋白質の遺伝子のみが濃縮されていく。このことにより特異的なRNA-蛋白質相互作用をとらえることが可能となると期待される。自らのmRNAに結合することがすでに知られている大腸菌リボソーム蛋白質S15を用いて、本システムの有効性を検討した。S15のmRNAの5'に存在するS15結合部位であるシュウドノット構造を形成できない変異型と、天然型の遺伝子をT7-tagと融合したものを作製した。この両者のmRNAを共存させたサンプルをPUREシステムで翻訳させ、合成された蛋白質に結合するmRNAをT7抗体により回収した。その結果、変異型に比べて天然型のRNAが優先的に選抜されていることが示された。現在この濃縮効率の向上を検討しているが、本システムが自らmRNAの結合するペプチドを選抜するのに有効なことが示された。

  • ゲノムの侵略と協調に関する総合的研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    1999年
    -
    2000年
     

    嶋 昭紘, 上田 卓也, 馳沢 盛一郎, 雨宮 昭南, 大矢 禎一, 河野 重行

     概要を見る

    昆虫のテロメラーゼとテロメア特異的レトロトランスポゾンを調べ、昆虫ゲノムの動的変動を明らかにした。出芽酵母の利己的遺伝子が自己伝播するために必要な宿主側と遺伝子内部の要因を明らかにした。植物マイコプラズマの各種変異株の染色体外DNAの遺伝子構造と複製様式・発現動態を解明した。メダカ培養細胞ではカスパーゼ活性がピリミジン2量体により誘導されることを明らかにした。tRNAのアミノ酸受容能の両義性による多義語コドンの発生メカニズムを、in vitroでの変異tRNAの機能解析から解明した。HNI系統メダカの生殖細胞自然突然変異率を調べ、優性致死突然変異を伴った総突然変異率では雌が桁違いに高いが、生存突然変異率では雄が高いことと、広範囲にわたるゲノム欠失を伴う雌ゲノムの変異が経世代的に伝播しにくいことを明らかにした。ヒロハノマンテマを用いて植物Y染色体に特異的なSTSマーカーを作成、一花粉解析法の確立、さらにBACライブラリーを構築してSTSマーカーを用いて,約100kbpのY染色体特異的DNA配列を単離した。GFP-tubulin遺伝子導入株などを用いて、植物細胞の形態形成、形態制御における表層微小管の機能解析を行った。トリノアシ卵の人工受精に始めて成功し、着床に至までの発生を記録、さらにトリノアシのHox,Otx,engrailed遺伝子の分離に成功し、発現パターン解析を行った。ゼブラフィッシュと霊長類の視物質遺伝子に新規レパートリーを発見した。高等植物とシアノバクテリアでの各種の環境ストレスに対する応答メカニズムを解明した。ホメオボックスジーンCdx2で形質転換した小腸陰窩細胞IEC-6を作製し、上皮細胞化を検討した。エクジソン分泌を抑制する前胸腺抑制ペプチド(PTSP)の単離、構造決定、産生器官同定を行った。神経幹細胞の細胞応答が、ガン抑制因子によって制御されていることを見つけた。嗅覚受容体が匂いを認識する分子機構を再構成系を用いて解明した。

  • 複雑系としての蛋白質合成系

    日本学術振興会  科学研究費助成事業

    研究期間:

    1999年
    -
    2000年
     

    上田 卓也, 鈴木 勉, 大矢 禎一

     概要を見る

    我々はCandida酵母においてCUGコドンがセリンとロイシンの2種のアミノ酸を同時に指定していることを、tRNAの解析と遺伝学的な手法により明らかにした。この多義的な遺伝暗号変換機構のCandida酵母における生物学的意義を解明するために、多義語コドンを司るtRNASerCAGのロイシン受容能を定量的に変化させ、細胞内あるいは生育への影響を解析しようとしている。これまでの解析によりtRNA^<Ser>CAGのロイシン受容能はアンチコドン3'側隣接塩基である37位の1-メチルグアノシン(mlG)のメチル基によって支配されていることが明らかになっている。今回はさらに受容能を向上させるために、73位の識別部位をGからAに改変することを試みた。細胞内に変異tRNAを発現させる前に、in vitroでロイシン受容能を評価するために、T7RNApoIymeraseを用いた未修飾tRNAを作製したがロイシンの取り込みは全く検出されなかった。さらに、今回新たに大腸菌m^lG methylaseを組換えタンパク質として調製し、未修飾tRNAの37位をメチル化したところ、nativeなものとほぼ同等のロイシン受容能が確認された。この結果は分子内のたった一つのメチル基がアミノアシルtRNA合成酵素の決定因子になりうることを直接的に示した初めての例である。さらに、73位の識別部位をAに置換すると、ロイシン受容能は飛躍的に向上しnativeなtRNA^<Ser>CAGをはるかに凌ぐ活性が確認された。Kcat/Kmの値としては、識別塩基がAへの変異は400倍の上昇を、37位をGからm^lGにしたtRNAでは、10倍の上昇が観察された。これらの結果を基に、細胞内でこれらの変異型tRNAを導入し、細胞機能の変化を観察する予定である。

  • リボソームRNAの機能構造解析

    日本学術振興会  科学研究費助成事業

    研究期間:

    1998年
    -
    2000年
     

    渡辺 公綱, 鈴木 勉, 上田 卓世, 新田 至, NIERHAUS K.H, NOLLER H.F.

     概要を見る

    本研究はリボソームとリボソームRNA(rRNA)の機能構造を解析することにより、生命体の最も基本的なシステムである翻訳系が如何に機能し、如何に構成されているかの根本原理を解明し、ひいてはそれが如何に生じたかを推論するための基礎的な知見を得ることを目的に立案されたものである。
    最初は大腸菌リボソームとrRNAの機能構造を解析するため、大腸菌リボソームをピリジンで処理し、部分的に蛋白質の剥稚したリボソームを用いて翻訳反応の素過程を探る研究を行なった。その結果、ピリジン存在下でも非酵素的(自発的)な転位反応が起り得ることが分かった。
    しかし一昨年当たりからリボソームそのものの結晶のX線構造解析が有用なデータを提供し始め、リボソーム上の翻訳反応の素過程もこの原子レベルの高次構造からあらかた推定可能なことが判明したため、研究のターゲットを哺乳動物ミトコンドリアに移し、初期の目的の達成を目指すことにした。このミトコンドリア・リボソーム(ミトリボソーム)は大腸菌のものと比較して、RNAが約半分に短縮されている代わりに、蛋白質が肥大化し、全体として分子量、サイズとも同程度になっているという特徴がある。翻訳機能を担うRNAが短縮しているため、機能部位の同定が容易であり、かつ短縮したRNAに結合する蛋白質は肥大化しているため、RNAから蛋白質への構造的、概能的な役割譲渡の原理解明の可能性がある。現在プロテオーム解析手段を用いて約70種類の蛋白質のうち大腸菌の対応する蛋白質と比較することにより55種類の同定に成功し、そららのcharacterizationを行なっている。またL7/L12蛋白質を大腸菌のもので置き換えたハイブリッド型ミトリボソームは、ミトコンドリアと大腸菌両方のtRNA、翻訳因子を受け入れ翻訳反応を行なうことができる。この互換性の由来をも解明しつつある。

  • 動物ミトコンドリアの翻訳システムの分子機構

    日本学術振興会  科学研究費助成事業

    研究期間:

    1996年
    -
    1998年
     

    渡辺 公綱, 新田 至, 河合 剛太, 上田 卓也

     概要を見る

    本基盤研究の目的は、(1)動物ミトコンドリアtRNAの構造解析と、(2)ウシ・ミトコンドリアのイン・ビトロ翻訳系の構築によるこれらのtRNAの翻訳活性の検証を通して、動物ミトコンドリア(mt)の翻訳システムの分子機構を解明することである。その翻訳システムは、原核細胞や真核細胞の細胞質におけるシステムとはかけ離れた顕著な特徴(異常な2次構造をもつtRNAと変則暗号の存在、tRNAが22種類しか存在しない、開始と伸長兼用のtRNA^<Met>、リボソームはRNAが短く、蛋白が多い、など)を持つことが主としてmtゲノムの塩基配列の解析から推定されていた。我々は翻訳システムの個々の構成成分を単離し、characterizationすることにより、以下の知見を得た。
    1) mt・tRNAの変則暗号解読機構ーー種々の生物のmtで変則暗号を見出し、それらを解読するtRNAを構造解析した結果、主としてアンチコドン1字目(Wobble位)の塩基(多くは修飾塩基)により暗号変化が引き起こされることを突き止め、ミトコンドリア独自のWobble則を確立した。
    2) ミトコンドリアtRNAの機能構造ーー生化学実験と^1H-NMR測定を組み合わせて、DアームやTアームを欠く異常な2次構造をもつtRNAでもL型に類似した立体構造を取り得ることを立証した。
    3) in-vitro翻訳系を用いた暗号解読機構の検証ーーmt成分のみからなるin-vitro翻訳系を構築し、これを用いてAUA(イソロイシン)コドンがf^5Cという修飾塩基をWobble位に持つtRNA^<Met>によってメチオニンに翻訳されることを実証した。
    4) アミノアシルtRNA合成酵素、ペプチド鎖伸長因子、解離因子ホルミルトランスフェラーゼ等の因子類単離精製し、キャラクタリゼーションし、大腸菌での大量生産系を確立した。それら因子類とtRNAとの相互認識機構を解析した。

  • RNAワールドからRNPワールドへ

    日本学術振興会  科学研究費助成事業

    研究期間:

    1996年
    -
    1997年
     

    上田 卓也

     概要を見る

    生命に誕生において、まずRNAのみからなるRNAワールドが原始地球上に存在し、その後このRNAワールドに蛋白質が取り込まれたするシナリオが有力なものになりつつある。しかし、この蛋白質の取り込みについての道筋に関してはなんら説得力のある仮説は提唱されていない。本研究は、このプロセスについて、現在の生物の分子機構の解明を基盤としてアプローチした。
    蛋白質の合成は、現在の生物では、巨大な蛋白質-RNA複合体であるリボソームで行われるが、そのペプチド鎖形成反応を行う分子(ペプチジルトランスフェラーゼ)が、リボソーム蛋白質であるのか、リボソームRNAであるのかは、未だ不明である。1992年に、H.Noller(米)のグループは、除タンパク処理を行ったリボソームでもペプチド鎖形成反応をすることを見いだしたが、この除タンパク処理は不十分であり、RNAがペプチジルトランスフェラーゼ活性を持つという結論には到達できなかった。本研究では、まずリボソームRNAを試験管内での転写反応によって合成し、完全に蛋白質のないシステムを構築した。このシステムを用いて、様々な条件を検討した。その結果、23SリボソームRNAの構造を組み直すフォールディング処理を行い、また界面活性剤であるSDSを添加することにより、ペプチジルトランスフェラーゼ活性を発現させることに成功した。この結果は、リボソームRNAがペプチジルトランスフェラーゼそのものであることを示す最初の実験的証拠となった。また,リボソームRNAの蛋白質合成活性の発現の反応機構を詳細に検討した。その結果,リボソーム同様に二つ以上のtRNA結合部位を持つこと,またtRNAはtを適切な位置に固定することにより,自発的な反応を引き起こすことを,明らかにした。これらの研究は,RNAワールドから蛋白質合成系が誕生する過程を明らかにする上で,重要な知見となるものと期待される。

  • リボゾーム機能とユウキ触媒活性の融合による有用蛋白質の生産

    日本学術振興会  科学研究費助成事業

    研究期間:

    1996年
    -
    1997年
     

    渡辺 君綱, 大部 良隆, 志賀 昭信, 新田 至, 上田 卓也

     概要を見る

    生細胞中でのタンパク質合成反応はリポソームと呼ばれる細胞内小顆粒上で進行する。そして、リポゾーム上で合成されるポリアミノ酸すなわちタンパク質は、メッセンジャーRNA(mRNA)にコードされた遺伝情報に従って、分子量やモノマー配列といった一次構造が厳密に制御されており、その一次配列の均一性によって高機能性を発揮する。本研究では、生物由来のタンパク質合成システムを基盤として、天然に存在する20種類のアミノ酸しか使用できないという生物独自の制約に縛られない、新たなバイオシステムの創製を行う。さらに、このシステムを利用して材料工学的に興味深いにもかかわらず従来技術では生産不可能であった機能性タンパク質の合成を試みる。
    具体的には、リポゾームを中心としたタンパク質合成系を、有機溶媒の一つであるピリジンと水との混合系中で再構築する。従来利用されてきた水溶液中での生体外タンパク質合成系は、リポゾームやmRNA以外に多数の可溶性タンパク質因子や化学エネルギー源であるアデノシン三リン酸(ATP)およびグアノシン三リン酸(GTP)を必要とし、モノマーであるアミノ酸をアミノアシル-tRNAの形で活性化してこの系に加えることにより、アミノ酸をmRNA特異的に重合している。これらの各成分を調製・最適化して安定な「タンパク質の生産システム」を構築することは極めて困難であり、実用化にはほど遠いのが現状である。この問題を解決する有望な手段として、我々は50-60%のピリジンを添加することにより、可溶性タンパク質因子やエネルギー源を一切必要とすることなく、アミノアシル結合のエネルギーのみで駆動する、新規な生体外タンパク質合成系の開発に成功した。

  • 変異リボトキシンを用いたリボソーム暗号解読中心の研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    1996年
    -
    1997年
     

    正木 春彦, 上田 卓也

     概要を見る

    1.コリシンE3,E4,E6のC末端ドメインE3C,E4C,E6Cと,E3Cの活性中心変異体(E517Q),およびそれぞれのインヒビターImmE3,ImmE4,ImmE6と,E3耐性を獲得したImmE6点変異体(W47C)を精製し,各Cドメインのリボソーム失活活性,リボソームの結合特性,Immとの結合特性といった分子レベルでの特異性を,各imm遺伝子の各コリシンに対する耐性度(免疫性)という生物学的特異性と対照させた.まず,E4CとE6Cはいずれもリボソームに対しE3Cと同一の作用様式を持つことを明らかにした.さらにC/Immの解離平衡定数Kdが10^<-8>M以上ではコリシン感受性となり,効果的な1アミノ酸置換でKdは1桁小さくなって辛うじて耐性を獲得すること,この閾値は,Immと結合が競争関係にあるリボソームとCドメインとのKdが規定していること,またKdは結合ではなく解離の速度定数だけで決定されていることが判明し,正しい複合体では大きな構造変化の起こっている可能性が考えられた.
    2.NMRによりE6CとImmE6の立体構造を決定した.E6Cは逆平行5本鎖βシートを骨格とし,β3-β4上に活性中心があり,シートに対して裏側のβ1-β2のループの付け根に特異性決定基がある.ImmE6は先に決定していたImmE3に似て,逆平行4本鎖βシートとループから成り,特異性決定基はシートの端に存在していた.chemical-shift perturbationでE6C,ImmE6両分子の結合面を同定した.さらに遺伝学的に推定していた特異性決定基,E6CのA496とImmE6のW47間に分子間NOEを観測し,これらが直接相互作用していることがわかった.また遊離のImmE6分子上では,二つの特異性決定基H5とW47間に側鎖間NOEを見出し,協同性が推定された.

  • タンパク質合成系の分子解剖

    日本学術振興会  科学研究費助成事業

    研究期間:

    1995年
    -
    1996年
     

    渡辺 公綱, 新田 至, 上田 卓也, HARRY Noller, KNUD Nierhau, NOLLER F.Har, NIERHAUS H.K, HIERHAUS Knu

     概要を見る

    分子生物学は、核酸塩基間の水素結合による相補性の発見を基礎とした、DNAの二重螺旋構造モデルに提唱により誕生した。このモデルを基礎として、核酸に蓄えられた遺伝情報がタンパク質として発現される構造が解明されたが、1980年代のリボザイムの発見によって、核酸、特にRNAの触媒活性という新たな側面が注目されるようになってきた。このような状況下で、我々は、翻訳因子やエネルギー源を用いなくとも、ピリジン、さらにプリンおよびピリミジン塩基類が、リボソーム上でのタンパク質合成を活性化するという現象を発見したが、この発見は、原始タンパク質合成系のアミノ酸重合反応において、核酸の塩基部分に存在する芳香族性三級アミンが触媒として重要な役割を演じていた可能性を示唆している。
    リボソームは数十種類のリボソームタンパク質と数種類のリボソームRNA(rRNA)から構成される大、小サブユニットの会合体である。本協同研究先であるドイツ・マックス・プランク研究所Nierhaus教授は、このリボソームを各構成要素に分子解剖し、必要最小要素のみを用いて再構成する技術に関する世界的権威であり、リボソームの構成要素の一つであるrRNAに作用する抗生物質や、tRNAのリボソームに対する結合様式に関する研究についても先端的な研究を展開している。本研究は我々の芳香族性三級アミンに関する実験技術と、Nierhaus教授のリボソーム分子解剖術を融合することにより、単純化されたリボソーム、特にリボソームRNAを用いて、タンパク質合成系におけるこれら塩基類の触媒メカニズムを詳細に検討し、タンパク質合成系の起源を解明することが目的であった。
    平成7年度中に、タンパク質合成系の最適化、およびリボソームの解体条件を確立した。すなわち、最適三級アミン化合物を選定し、反応温度、三級アミン化合物濃度、カチオン濃度を最適化した。特に合成産物であるタンパク質の分子量や、鋳型の翻訳精度を確認した。その結果、合成産物がポリフェニルアラニンの場合で重合度は少なくとも5以上、ポリリジンで40であること、さらにウリジル酸とシチジル酸の交互共重合体を鋳型とした場合の生成産物はセリンとロイシンの交互共重合体で、生成産物の重合度および翻訳精度のいずれも、十分実用に耐え得るものであることを確認した。さらに、リボソーム解体の条件をNierhaus教授の指導の下に確立した。その結果、rRNAの大量調製が可能となった。
    またrRNAのみでもタンパク質合成活性が存在することが明らかとなった。タンパク質合成の中心的触媒であるリボソームはリボソームタンパク質とrRNAから構成されていることは既に述べた所であるが、そのいずれにタンパク質合成の活性中心が存在するかは、長い間議論されてきた。ここで得られた知見により、タンパク質合成の活性中心はrRNAに存在していることが証明された。
    平成8年度は、試験管内転写により調製されたrRNAを用いて以下の実験を行い、rRNAの活性中心部位を明らかにした。
    (1)三級アミンの作用部位に変異を導入したrRNAを作製、またその一部をNierhaus教授より供与していただき、それらの活性を検討した。その結果、rRNAのドメインVに存在する中央ループ近傍がペプチド結合生成活性に直接関与していることが判明した。
    (2)rRNAに作用する抗生物質(クロラムフェニコールなど)や毒素(RIP)を用いることにより、活性中心を検討した。その結果、rRNAのドメインVIに存在するαサルシンループが、ペプチジルtRNA移動反応に直接関与していることが判明した。
    (3)非酵素的tRNA結合実験によりrRNA上のtRNA結合部位を明らかにすることを目的とし、非酵素的tRNA結合条件を確立した。
    以上に述べたように、本協同研究は極めて順調に成果をあげることができた。今後は、断片化されたrRNAを用いることにより、活性発現に必要な最小断片を同定するなど、さらに研究を進めていく予定である。

  • 酵母における遺伝暗号変化の分子機構

    日本学術振興会  科学研究費助成事業

    研究期間:

    1995年
     
     
     

    上田 卓也

     概要を見る

    一群のCandida酵母は通常ロイシンのコドンであるCUGをセリンに翻訳する。私はこの暗号変化がCUGコドンをセリンに翻訳する特殊なセリンtRNA(tRNA^<Ser>CAG)の出現によるものであることを9種類のCandida酵母においてすでに明らかにしている。
    C.zeylanoidesをはじめとするこれらのtRNAは、in vitroにおいてセリンを効率よく受容するが、アンチコドンループにロイシンtRNAの特徴を有しているために、若干のロイシン受容能を持つこと、またその認識機構を昨年度報告した。今年度は、in vitroにおいて観測されたセリンtRNAのロイシル化が、in vivoで実際に生じているかについて焦点をあてた。まず細胞内において、tRNA^<Ser>CAGがロイシンを受容しているかを確認するために、細胞内アミノアシルtRNAの分析法を確立した。この手法により、C.zeylanoidesのtRNA^<Ser>CAGが細胞内でセリンに対し約3%のロイシンを実際に受容していることが明らかになった。次に、このロイシル化したセリンtRNAが、タンパク質合成に参加して、CTGコドンにセリンと若干のロイシンを取り込むかを示すため、C.maltosaのura3変異株を用い、活性に必須な45番目のロイシン残基がCTGコドンでコードされているS.cerevisiae由来のURA3遺伝子を導入して相補を試みた。コントロールとして対応する位置をTCTコドンに改変した遺伝子では全くの相補が観測されなかったのに対し、CTGコドンでは、若干の相補が観測された。この結果は、CTGコドンの若干のロイシル化に由来するURA3遺伝子の低レベルの活性発現によるものであると考えられる。したがって、CTGコドンはセリンとロイシンに翻訳されると結論できる。この知見は1つのコドンが1つのアミノ酸に対応するというこれまでの常識を覆すものであり、遺伝暗号の二重指定状態(dual assignment)であると考えている。二重指定状態が実際細胞において不可欠であるかどうかは、今後の課題であるが、暗号変化の中間状態を支える重要な役割を果たしたと考えている。

  • 酵素における遺伝暗号変化の分子機構

    日本学術振興会  科学研究費助成事業

    研究期間:

    1994年
     
     
     

    上田 卓也

     概要を見る

    一群のカンディダ酵母の核の遺伝子発現系においてCUGコドンがロイシンではなくセリンに翻訳される特異な変則暗号を使用している。さらに5SrRNAなどを用いた分子系統学的研究から、この酵母の世界においてCUGコドンに対応するアミノ酸がロイシン→セリン→ロイシンと二度変化していることを明らかにした。またこの変則暗号の解読を行うセリンtRNAを,これらのカンディダ酵母から単離し一次構造解析を報告した。この解析結果から、CUGに対応するセリンtRNAは通常のパン酵母のセリンtRNAとは違ったいくつかのユニークな特徴を持つ。その中で,最も興味深い点は、アンチコドンの5'側の塩基(33位)がUではない点である。開始tRNA以外の既知のtRNAでは,この部位は例外なくUであるのに対して,この変則暗号に特異的なセリンtRNAはG(一例のみC)をこの位置に持つ。また、CUGコドンを多様するC.cylindraceaではアンチコドンの3'隣接塩基は末修飾のAである(これはセリンtRNAの特徴)が、それ以外ではロイシンのtRNAと同様にm^1Gである。こうしたアンチコドンの特異な構造がこの変則暗号の発生と普遍暗号への復帰の過程に,何らかの役割を果たしたと可能性が高い。この点を明らかにするために,機能と構造の二面からアプローチしている。
    まずこの33位がUではなくGになることでロイシルtRNA合成酵素の誤った結合を防止するための,負の決定因子の一つとして機能しているのではないかと考え、次のような実験を行った。C.zeylanoidesのtRNAの33を分子整形によって他の塩基に置換し、ロイシルtRNA合成酵素によるミスチャージの程度を測定した。その結果,U,Cに置換したものではミスチャージが上昇した。それに対して、Aに置換したものでは減少した。この結果は、酵母のロイシルtRNA合成酵素はアンチコドン領域を認識していることが示された。しかし、G33であることによってミスチャージを完全に防止しているとは結論できなかった。この点については、現在T7転写系での変異RNAを作製して詳細に解析しつつある。また37位の塩基の役割については、C.cylindraceaのtRNA(Aを持つ)と、それ以外のm^1GをもつtRNAのロイシル化を検討したところ、Aであることによってミスチャージが強く押さえられることが明らかになった。つまり、負の決定因子である可能性が高いことが明らかになった。

  • カンジダ酵母における遺伝暗号変化の分子進化学的研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    1993年
     
     
     

    上田 卓也

     概要を見る

    Candida cylindraceaの遺伝暗号表ではコドンCUGはセリンに対応する。しかしCURのもう一方のコドンであるCUAについては,アミノ酸の対応は不明である。CUNのコドンボックスに対応するロイシンのtRNAとしては二種類単離されているがアンチコドンはともにIAGであり,通常のwobble ruleに従えば大腸菌などの原核生物と同様にイノシンがAも読み、CUAはロイシンとなるはずである。しかし,Munzらの遺伝学的解析から,真核生物ではイノシンはAを読めないことが定説となっている。また、ではイノシンではなく修飾されたUがAに対応するとされている。二つの可能性を検討するためにCandida cylindraceaの無細胞たんぱく質合成系を構築し,CUAを含む合成mRNAを用いてCUAの対応するアミノ酸を決定した。その結果Candida cylindraceaの全tRNA画分を用いた場合,CUAはロイシンに翻訳され,Candida cylindraceaにおいてCUAはロイシンであった。また、精製したIAGのアンチコドンを持つロイシンtRNAを用いても,CUAコドンがロイシンとして翻訳された。従って,酵母においてもイノシンはAともリボソーム上で塩基対を形成すると考えべきである。
    すでに5SrRNAの配列に基づきカンジダ酵母の系統関係を作製し,CUGコドンがロイシン(グループI)→セリン(グループII)→ロイシン(グループIII)の経路で変化したことを示す結果を我々は得ている。本年度はEF-1αの塩基配列を決定し,それに基づく系統樹を作製した結果,このプロセスの正当性が確認された。CUNボックスに対応するtRNAのアンチコドンの一文字目はグループIとIIのものがIとCであるが,それに対してグループIIIに属するSaccharomyces cerevisaeでは未修飾のUのものしか存在しない.この未修飾のUの存在は遺伝暗号の変化のなごりと考えられる。

  • 非天然アミノ酸のタンパク質への組み込み

    日本学術振興会  科学研究費助成事業

    研究期間:

    1992年
    -
    1993年
     

    西川 一八, 石川 正英, 上田 卓也

     概要を見る

    1)非天然型のアミノアシルpCpCpAの化学合成を行う際、保護基の選び方が単離収率に大きく影響することがわかったので、5′末端のリン酸および該酸塩基のアミノ基の保護基として、それぞれ中性条件下パラジウム錯体を用いて均一系で除去できるアリル基およびアリルオキシカルボニル基を導入し、インターヌクレオチドのリン酸と核酸の2′水酸基の保護基にはそれぞれアリル基およびエトキシエチル基を用いて合成を行った。非天然アミノ酸としては、タンパク質への組み込み後の検出を容易に行えるようにするため、リシンの側鎖にビオチン基を導入したビオチニルリシルpCpAを合成した。
    2)酵母フェニルアラニンtRNAやチロシンtRNAなどを出発材料に、RNAの分子整形術を駆使してアンチコドンに任意の配列のトリプレットを持ち、3′末端のpCpCpAを欠くtRNAを大量に作製できるようにした。また、ビオチン修飾したRNAやタンパク質を高感度に検出する方法の検討を行い、放射性標識を用いずにRNAの塩基配列を解析できる新しい方法を開発した。
    3)大腸菌の無細胞タンパク質合成系とチオ-mRNAを用いたモデル実験系を構築し、チオ-mRNAがヌクレアーゼ耐性や翻訳効率の点で天然のmRNAよりも優れていることを示した。また、高度好熱菌の無細胞タンパク質合成系におけるポリアミンの影響について検討した。さらに酵母や牛ミトコンドリア由来の無細胞系についてもタンパク質合成の安定化について基礎的な条件等を検討した。

  • In vitro 翻訳系の調製によるミトコンドリアの暗号解読機構の研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    1991年
    -
    1993年
     

    渡辺 公綱, 横川 隆志, 河合 剛太, 上田 卓也, 西川 一八, SPREMULLI Li, LINDA Lucy S, SPREMULL Lin

     概要を見る

    本国際学術共同研究は、動物ミトコンドリアにおける暗号変化(UGA;普遍暗号では終止暗号がトリプトファンに、AUA;イソロイシンがメチオニンの暗号に、AGA/AGG;アルギニンが殆どの無脊椎動物ではセリン、原索動物ではグリシン、脊椎動物では終止暗号に変化、など)の分子機構をin vitro翻訳系を構築して、解明する目的で始められた。このような研究は、ミトコンドリア(mt)からその細胞内量から見ても、生化学的な研究に十分な試料を調製することが大変困難なこと、翻訳に関わるタンパク性諸因子がかなり不安定で単離が困難なことなどが主な障害となって世界的にも殆ど手がつけられていなかった。我々は特異構造を持つmt・tRNA(殆どのmt・tRNAではL型立体構造形成に関わっているDループとTループ間の塩基対を欠いていたり、DループやTループが欠落したものも見つかっている)と変則暗号解読の因果関係を探る目的で、mt・tRNAの大量調製法を確立し、その構造と性質を調べていたが、翻訳系の構築に必要な活性のある因子の調製ができなかった。国外共同研究者であるSpremulliのグループは、活性あるmtリボソームと翻訳系諸因子の調製に成功していたが、mt・tRNAの単離ができなかった。このような状況においてお互いのグループで開発したシステムと技術を合体させることにより、mtのinvitro翻訳系を構築し、暗号変化の分子機構を解明するという目的で平成3年度から本研究がスタートした。研究はかなり順調に進んできたが、本格的な展開はこれからであり、やっとその基礎が固まったという現状である。以下に年度を追ってその成果を述べる。
    [平成3年度]
    1)tRNAの特定配列に相補的な合成DNAプローブを用いたハイブリダイゼーション法を開発し、mt・tRNAの0.2-0.5mgオーダーの調製が可能になった。
    2)UCN(N=A,U,G,C)のコドンに対応するウシmt・セリンtRNAを単離、精製し、それが従来の遺伝子から推定されていた配列から、実際の構造がずれていること、アンチコドン・ステムは一塩基対長く、アミノ酸ステムとDステムの間が一塩基しかない、異常な2次構造をとること、この構造は哺乳動物mtに共通であることを明らかにした。
    3)ウシ肝臓から活性のあるリボソーム、開始因子(IF-2)、伸長因子(EF-Tu/Ts、EF-G)、アミノアシル-tRNA合成酵素(ARS)の調製方法を確立し、それらの性質を検討した。
    [平成4年度]
    1)AGY(Y=U,C)のコドンに対応する、Dアームを欠くセリンtRNAのセリルtRNA合成酵素(SerRS)による認識部位を決定する目的で、このtRNA遺伝子からT7RNAポリメラーゼによる転写物を調製し、種々の塩基置換を導入したtRNA変異体のSerRSによるセリン受容能を測定した結果、アンチコドンは認識に無関係だが、Tループが重要であり、中でもよく保存されたループ中央のA44の置換が決定的であることが分かった。
    2)ウシ・mtでポリ(U)依存ポリ(フェニルアラニン)合成系を初めて構築し、大腸菌の系との構成成分の互換性を検討したところ、mtのPhe-tRNA^<Phe>は大腸菌のEF-TuとGTPとで3者複合体を形成するが、そこからリボソームA部位への転移過程が働かないことを明らかにした。
    3)ウシ・mtのメチオニンtRNAの塩基配列を再検討し、アンチコドンの一字目に、5-ホルミルシチジンという新規修飾塩基が存在することを明らかにした。
    4)ウシ・mtフェニルアラニンtRNAの修飾塩基を含む塩基配列を決定し、RNaseや化学試薬への感受性からその立体構造を推定したところ、Dループ、Tループ相互作用はないが、Dアームとバリアブルループ間の3次元的な塩基対形成によってL型に近い構造をとっていることを見出した。
    [平成5年度]
    1)ポリ(U)依存ポリ(Phe)合成系の効率化の条件を検討し、1mMスペルミン存在下で大腸菌の系の約1/2のレベルまで合成効率を上昇させることに成功した。
    2)AUAがメチオニンの暗号であることを証明するために、AUAを含む人工mRNAを用いて、AUAに依存したメチオニル-tRNAのリボソームへの結合、ポリペプチドへのメチオニンの取り込みを調べたが、現在までのところまだ肯定的な結果は得られていない。
    3)ウシmtからホルミルトランスフェラーゼを精製し、fMet-tRNAを作成し、EF-TuとIF-2の結合をMet-tRNAと比較したところ、fMet-tRNAはIF-2と、Met-tRNAはEF-Tuとそれぞれより高い親和性を示した。これは単一tRNAがホルミル化によって開始と伸長の両反応に使い分けられる可能性を支持するものである。

  • 原索動物ミトコンドリアの変則暗号とtRNA

    日本学術振興会  科学研究費助成事業

    研究期間:

    1992年
     
     
     

    上田 卓也

  • カンジダ酵母における遺伝暗号変化の分子進化学的研究

    日本学術振興会  科学研究費助成事業

    研究期間:

    1992年
     
     
     

    上田 卓也

     概要を見る

    1989年川口らはカンジダ酵母でるCandida cylindraceaのlipaseIの遺伝子で普遍暗号ではロイシンであるCUGがセリンに変化していることを発見した。我々は変則暗号の成立メカニズムを探る上で良い材料と考え、Candida cylindraceaのCUGが解読するセリンtRNAとその遺伝子を構造解析を行なった。その解析結果から、この遺伝子がアンチコドン部分にイントロンをもち、この事によってUCN系のセリンのtRNAから一回の変化によってCUGコドン対応できるように変化した進化の経路が示唆された。またCandida cylindraceaのIipaseIの遺伝子の偽遺伝子の配列を解析しセリンの部位を比較したところUCGがCUGに変化している偽遺伝子を見いだした。このことは変則暗号UCGがUCGから由来である考え方が妥当であることを示している。
    さらに大沢研究室と共同研究の結果、カンジダ酵母でCandida cylindracea以外の5種類の株でこの変則暗号が使用されていることを明らかにした。またこれらの株を5SリボソームRNAの配列から系統樹を作製し、酵母の中でCUGがロイシン→セリン→ロイシンの経路で変化していることを明らかにした。また変則的なCUGをセリンに使用する株の一つであるCandida zeylanoidesのゲノム上に、このtRNA遺伝子と相同性が高く恐らく同一のtRNA遺伝子を起源とすると思われるセリンのtRNA遺伝子を発見した。このセリンのtRNAはUCGのコドンに対応するtRNAであり、我々のセリンtRNA由来であると言う考え方を強く支持した。

  • 動物細胞ミトコンドリアに存在するプロセスされた5SrRNAの構造と機能

    日本学術振興会  科学研究費助成事業

    研究期間:

    1991年
     
     
     

    上田 卓也

  • 遺伝暗号の多様性

    日本学術振興会  科学研究費助成事業

    研究期間:

    1989年
    -
    1991年
     

    渡辺 公綱, 上田 卓也, 田仲 可昌, 口野 嘉幸, 大山 莞爾, 大澤 省三

     概要を見る

    遺伝暗号の可変性は確立された概念となったが、本研究班では、各生物、オルガネラにおける遺伝暗号の変化とその範囲を明らかにし、過去から現在にいたる変遷の過程を知ることにより、暗号多様化の実体とそのメカニズムを探り、多様化の要因を解明することを目的としてきた。新たに見つかったカンジダ酵母の暗号変化(CUG,Leu→Ser)について、共同研究が行われ、CUGをSerに翻訳するtRNAの構造解析(上田、渡辺、口野)と、その翻訳活性の検出(大浜、大沢)に成功した。5SRNAを指標とした各種不完全菌類の系統関係と翻訳系との対応も調べられている(大浜、大沢)。遺伝暗号の多様な変化が見つかっているミトコンドリアでは、まだ解析が行われていない種々の動植物について、ミトコンドリア遺伝子中のコドンとtRNAアンチコドンの両面の解析が行われた。新たな暗号変化としてプラナリアでUAA(Stop→Tyr)(大浜、大沢)、ホヤでAGR(Arg→Ser)(渡辺、上田)が見つかったが、全塩基配列が決定されたゼ-ゴケ(大山)や全長54kbのうち25kbの配列が決定された細胞性粘菌(田仲)では普遍暗号、RNA editingの証拠も現在までは見出されていない。暗号変化のメカニズムを調べるために不可欠なミトコンドリアのin vitroタンパク質合成系もウシ・ミトコンドリアで初めてポリ(U)依存ポリ(Phe)合成系が構築された(渡辺、上田)。暗号変化の進化的要因をよく説明するコドン捕獲説(大沢)の妥当性がマイコプラズマで実験的に検証された(大沢、武藤、安達)。レトロウイルスの感染によって動物細胞でUAGサプレッサ-グルタミンtRNAの発現が昂進するメカニズムとして、この遺伝子の5'上流に特異的に結合する核タンパク質の存在が明らかにされた(口野)。マウスにおけるapoBmRNA editingが発生、加令などの生理的条件で制御されることが示された(樋口)。

  • 進化工学

  • 蛋白質合成系の研究

  • Study on the Protein synthesis

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Misc

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    伍 広明, 松林 英明, 長田 江里子, 上田 卓也

    日本生物工学会大会講演要旨集   67   232 - 232  2015年

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    Neuro-Oncology   16 ( suppl 2 ) ii102 - ii102  2014年09月

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    高橋俊太郎, 上田卓也, 岡畑惠雄

    日本RNA学会年会要旨集   14th   88  2012年07月

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    廣瀬敦, 高橋俊太郎, 上田卓也, 岡畑惠雄

    日本RNA学会年会要旨集   14th   201  2012年07月

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  • 大腸菌翻訳開始複合体形成における翻訳エンハンサー配列の効果

    秋山裕也, 高橋俊太郎, 清水義宏, 上田卓也, 岡畑恵雄

    日本RNA学会年会要旨集   12th   158  2010年07月

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    高橋俊太郎, 清水義宏, 上田卓也, 岡畑恵雄

    日本RNA学会年会要旨集   12th   42  2010年07月

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    末松卓真, 横堀伸一, 森田寛幸, 吉成茂夫, 上田卓也, 北潔, 竹内野乃, 渡邊洋一

    日本RNA学会年会要旨集   12th  2010年

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    高橋俊太郎, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

    日本化学会バイオテクノロジー部会シンポジウム講演要旨集   12th   50  2009年09月

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    日本RNA学会年会要旨集   11th   121  2009年07月

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    日本RNA学会年会要旨集   11th   90  2009年07月

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    日本化学会講演予稿集   89th ( 2 ) 1425  2009年03月

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    日本化学会講演予稿集   89th ( 2 ) 1311  2009年03月

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    日本分子生物学会年会講演要旨集   32nd ( Vol.4 ) 79  2009年

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    日本化学会バイオテクノロジー部会シンポジウム講演要旨集   11th   197  2008年09月

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    日本化学会バイオテクノロジー部会シンポジウム講演要旨集   11th   199  2008年09月

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    飯田匡章, 高橋俊太郎, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

    日本化学会講演予稿集   88th ( 2 ) 881  2008年03月

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    磯部英美, 高橋俊太郎, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

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    生化学     4T5-12  2008年

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    生体機能関連化学シンポジウム講演要旨集   22nd   204 - 205  2007年09月

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    生体機能関連化学シンポジウム講演要旨集   22nd   426 - 427  2007年09月

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    高橋俊太郎, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

    日本RNA学会年会要旨集   9th   88  2007年07月

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    日本RNA学会年会要旨集   9th   150  2007年07月

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    日本化学会講演予稿集   87th ( 2 ) 1304  2007年03月

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    高橋俊太郎, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

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    生化学     3P-0634  2007年

    J-GLOBAL

  • 翻訳伸長段階におけるリボソームおよびtRNAのダイナミクスと分子擬態

    清水義宏, 上田卓也

    実験医学   25   5 - 12  2007年

  • 柔軟な翻訳開始システムの動力学的アプローチ

    高橋俊太郎, 秋田涼子, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

    日本化学会バイオテクノロジー部会シンポジウム講演要旨集   9th   210  2006年09月

    J-GLOBAL

  • 水晶発振子を用いた大腸菌翻訳過程の解析:翻訳伸長過程の観察

    秋田涼子, 高橋俊太郎, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

    日本化学会講演予稿集   86th ( 2 ) 843  2006年03月

    J-GLOBAL

  • 水晶発振子を用いた大腸菌翻訳過程の解析:mRNAへのリボソームの結合特性

    高橋俊太郎, 秋田涼子, 古澤宏幸, 清水義宏, 上田卓也, 岡畑恵雄

    日本化学会講演予稿集   86th ( 2 ) 843  2006年03月

    J-GLOBAL

  • Interaction analysis for a chaperone and a signal recognition particle binding onto a ribosome by using quartz-crystal microbalance (QCM)

    FURUSAWA Hiroyuki, FURUSAWA Yuki, MITOMO Hideyuki, SHIGEMATSU Hideki, UEDA Takuya, OKAHATA Yoshio

    生化学   77 ( 8 ) 1023  2005年08月

    J-GLOBAL

  • 無細胞蛋白質合成系: 大腸菌由来完全再構成系PURESYSTEM

    清水義宏, 上田卓也

    蛋白質核酸酵素   49   1520 - 1526  2004年

  • 無細胞タンパク質合成系へのピュアアプローチ

    上田 卓也, 清水 義宏

    生物物理   生物物理43(1)、9-14 ( 1 ) 9 - 14  2003年

     概要を見る

    ゲノムにコードされたタンパク質の構造と機能の総合的研究は,ポストゲノム時代の生命科学の中心的な流れとなると考えられる.私たちは,こうした研究におけるネックとなっているタンパク質の調製プロセスをハイスループット化するために,精製した大腸菌の翻訳因子から再構成した無細胞遺伝子発現系を構築することに成功し,PUREシステムと名づけた.PUREシステムによるタンパク質生産法は,タンパク質の研究におけるプラットフォーム技術になるものと期待している.

    DOI CiNii

  • ピュアなタンパク質をつくるためのPUREシステム

    Bioベンチャー(羊土社)   Vol.2 №1(1・2月号)  2002年

  • 無細胞タンパク質合成システム

    実験医学(羊土社)   Vol.20 №14(増刊号)  2002年

  • 無細胞タンパク質合成系の新展開

    清水義宏, 上田卓也

    遺伝子医学   6 ( 2 ) 266 - 270  2002年

    CiNii

  • Cell-free translation reconstituted with purified components

    Y Shimizu, A Inoue, Y Tomari, T Suzuki, T Yokogawa, K Nishikawa, T Ueda

    NATURE BIOTECHNOLOGY   19 ( 8 ) 751 - 755  2001年08月

     概要を見る

    We have developed a protein-synthesizing system reconstituted from recombinant tagged protein factors purified to homogeneity. The system was able to produce protein at a rate of about 160 mug/ml/h in a batch mode without the need for any supplementary apparatus. The protein products were easily purified within 1 h using affinity chromatography to remove the tagged protein factors. Moreover, omission of a release factor allowed efficient incorporation of an unnatural amino acid using suppressor transfer RNA (tRNA).

    DOI PubMed CiNii

  • Structural compensation for the deficit of rRNA with proteins in the mammalian mitochondrial ribosome - Systematic analysis of protein components of the large ribosomal subunit from mammalian mitochondria

    T Suzuki, M Terasaki, C Takemoto-Hori, T Hanada, T Ueda, A Wada, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 24 ) 21724 - 21736  2001年06月

     概要を見る

    The mammalian mitochondrial ribosome (mitoribosome) is a highly protein-rich particle in which almost half of the rRNA contained in the bacterial ribosome is replaced with proteins. It is known that mitochondrial translation factors can function on both mitochondrial and Escherichia coli ribosomes, indicating that protein components in the mitoribosome compensate the reduced rRNA chain to make a bacteria-type ribosome, To elucidate the molecular basis of this compensation, we analyzed bovine mitoribosomal large subunit proteins; 31 proteins were identified including 15 newly identified proteins with their cDNA sequences from human and mouse. The results showed that the proteins with binding sites on rRNA shortened or lost in the mitoribosome were enlarged when compared with the E. coli counterparts; this suggests the structural compensation of the rRNA deficit by the enlarged proteins in the mitoribosome.

  • An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria

    T Ohtsuki, Y Watanabe, C Takemoto, G Kawai, T Ueda, K Kita, S Kojima, Y Kaziro, J Nyborg, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   276 ( 24 ) 21571 - 21577  2001年06月

     概要を見る

    We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans, Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu, Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs, This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.

    DOI PubMed CiNii

  • ほ乳動物ミトコンドリアリボソームのプロテオーム解析 RNAからタンパク質への機能的,構造的な役割の委譲

    鈴木勉, 寺崎真樹, 竹本千重, 花田孝雄, 上田卓也, 和田明, 渡辺公綱

    RNAミーティング   3rd  2001年

    J-GLOBAL

  • A pathogenic point mutation reduces stability of mitochondrial mutant tRNA(lle)

    T Yasukawa, N Hino, T Suzuki, K Watanabe, T Ueda, S Ohta

    NUCLEIC ACIDS RESEARCH   28 ( 19 ) 3779 - 3784  2000年10月

     概要を見る

    Point mutations in mitochondrial tRNA genes are responsible for individual subgroups of mitochondrial encephalomyopathies, We have recently reported that point mutations in the tRNA(Leu)(UUR) and tRNA(Lys) genes cause a defect in the normal modification at the first nucleotide of the anticodon, As part of a systematic analysis of pathogenic mutant mitochondrial tRNAs, we purified tRNA(lle) with a point mutation at nucleotide 4269 to determine its nucleotide sequence, including modified nucleotides, We found that, instead of causing a defect in the post-transcriptional modification, a pathogenic point mutation in the mitochondrial tRNA(lle) reduced the stability of the mutant tRNA molecule, resulting in a low steady-state level of aminoacyl-tRNA. The reduced stability was confirmed by examining the life-span of the mutant tRNA(lle) both in vitro and in vivo, as well as by monitoring its melting profile. Our finding indicates that the mutant tRNA(lle) itself is intrinsically unstable.

    DOI PubMed CiNii

  • The role of tightly bound ATP in Escherichia coli tRNA nucleotidyltransferase

    Y Tomari, T Suzuki, K Watanabe, T Ueda

    GENES TO CELLS   5 ( 9 ) 689 - 698  2000年09月

     概要を見る

    Background: The CCA-adding enzyme [ATP(CTP): tRNA nucleotidyltransferase (EC. 2.7.7.25)] catalyses the addition of the conserved CCA sequence to the 3'-terminus of tRNAs. All CCA-adding enzymes are classified into the nucleotidyltransferase superfamily. In the absence of ATP, the Escherichia coli CCA-adding enzyme displays anomalous poly(C) polymerase activity.
    Results: We show that CCA-adding enzyme over-expressed in E. coli exists in an ATP-bound form. The affinities of ATP and CTP towards the enzyme were estimated by several methods, and the dissociation constants for ATP and CTP were determined to be 6.3 and 188 mu m, respectively. AMP-incorporation terminated the nucleotidyltransferase reaction, while in the absence of ATP, the enzyme continued poly(C) polymerization. In the case of a tRNA substrate with a mutation in the T-loop region, normal CC was added at a much slower rate compared with the wild-type, but anomalous poly(C) polymerization occurred at the same rate as in the wild-type.
    Conclusion: Based on the findings outlined above, we concluded that the E. coli CCA-adding enzyme possesses at least two distinct nucleotide binding sites, one responsible for ATP binding and the other(s) for CTP binding. The addition of ATP from the tight ATP binding site terminates nucleotide incorporation, thus limiting poly(C) polymerization to CCA. It is also suggested that during anomalous poly(C) polymerization, tRNA translocates from the tRNA binding site upon the third C addition.

    DOI PubMed CiNii

  • Characterization and tRNA recognition of mammalian mitochondrial seryl-tRNA synthetase

    T Yokogawa, N Shimada, N Takeuchi, L Benkowski, T Suzuki, A Omori, T Ueda, K Nishikawa, LL Spremulli, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   275 ( 26 ) 19913 - 19920  2000年06月

     概要を見る

    Animal mitochondrial protein synthesis systems contain two serine tRNAs (tRNAs(Ser)) corresponding to the codons AGY and UCN, each possessing an unusual secondary structure; the former lacks the entire D arm, and the latter has a slightly different cloverleaf structure. To elucidate whether these two tRNAs(Ser) can be recognized by the single animal mitochondrial seryl-tRNA synthetase (mt SerRS), we purified mt SerRS from bovine liver 2400-fold and showed that it can aminoacylate both of them. Specific interaction between mt SerRS and either of the tRNAs(Ser) was also observed in a gel retardation assay. cDNA cloning of bovine mt SerRS revealed that the deduced amino acid sequence of the enzyme contains 518 amino acid residues. The cDNAs of human and mouse mt SerRS were obtained by reverse transcription-polymerase chain reaction and expressed sequence tag data base searches. Elaborate inspection of primary sequences of mammalian mt SerRSs revealed diversity in the N-terminal domain responsible for tRNA recognition, indicating that the recognition mechanism of mammalian mt SerRS differs considerably from that of its prokaryotic counterpart. In addition, the human mt SerRS gene was found to be located on chromosome 19q13.1, to which the autosomal deafness locus DFNA4 is mapped.

    DOI PubMed CiNii

  • Defect in modification at the anticodon wobble nucleotide of mitochondrial tRNA(Lys) with the MERRF encephalomyopathy pathogenic mutation

    T Yasukawa, T Suzuki, N Ishii, T Ueda, S Ohta, K Watanabe

    FEBS LETTERS   467 ( 2-3 ) 175 - 178  2000年02月

     概要を見る

    A mitochondrial tRNA(Lys) gene mutation at nucleotide position 8344 is responsible for the myoclonus epilepsy associated with ragged-red fibers (MERRF) subgroup of mitochondrial encephalomyopathies. Here, we show that normally modified uridine at the anticodon wobble position remains unmodified in the purified mutant tRNA(Lys). We hale reported a similar modification defect at the same position in two mutant mitochondrial tRNAs(Leu)(UUR) in another subgroup, mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), indicating this defect is common in the two hinds of tRNA molecules with the respective mutations of the two major mitochondrial encephalomyopathies. We therefore suggest the defect in the anticodon is responsible, through the translational process, for the pathogenesis of mitochondrial diseases. (C) 2000 Federation of European Biochemical Societies.

    DOI PubMed CiNii

  • Modification defect at anticodon wobble nucleotide of mitochondrial tRNAs(Leu)(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes

    T Yasukawa, T Suzuki, T Suzuki, T Ueda, S Ohta, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   275 ( 6 ) 4251 - 4257  2000年02月

     概要を見る

    The mitochondrial tRNA(Leu)(UUR) (R = A or Gr) gene possesses several hot spots for pathogenic mutations. A point:mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAs(Leu)(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAs(Leu)(UUR) versus uncharged tRNAs(Leu)(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNA(Leu)(UUR) at the first position of the anticodon, The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAsLeU(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.

    DOI PubMed CiNii

  • ほ乳動物ミトコンドリアリボソームのキャラクタリゼーション

    鈴木勉, 寺崎真樹, 竹本千重, NG C G, 梶浦章正, 花田孝雄, 和田明, 上田卓也, 渡辺公綱

    RNAミーティング   2nd  2000年

    J-GLOBAL

  • Complete DNA sequence of the mitochondrial genome of the ascidian Halocynthia roretxi (Chordata, Urochordata)

    S Yokobori, T Ueda, G Feldmaier-Fuchs, S Paabo, R Ueshima, A Kondow, K Nishikawa, K Watanabe

    GENETICS   153 ( 4 ) 1851 - 1862  1999年12月

     概要を見る

    The complete nucleotide sequence of the 14,771-bp-long mitochondrial (rnt) DNA of a urochordate (Chordata)-the ascidian Halocynthia: roretzi-was determined. All the Halocynthia mt-genes were found to be located on a single strand, which is rich in T and G rather than in A and C. Like nematode and Mytilus edulis mtDNAs, that of Halocynthia encodes no ATP synthetase subunit 8 gene. However, it does encode an additional tRNA gene for glycine (anticodon TCT) that enables Halocynthia mitochondria to use AGA and AGG codons for glycine. The mtDNA carries an unusual tRNA(Met) gene with a TAT anticodon instead of the usual tRNA(CAT)(Met) gene. As in other metazoan mtDNAs, there is not any long noncoding region. The gene order of Halocynthia mtDNA is completely different from that of vertebrate mtDNAs except for tRNA(His)-tRNA(GCU)(Ser), suggesting that evolutionary change in the mt-gene structure is much accelerated in the urochordate line compared with that in vertebrates. The amino acid sequences of Halocynthia mt-proteins deduced from their gene sequences are quite different from those in other metazoans, indicating that the substitution rate in Halocynthia mt-protein genes is also accelerated.

  • Codon reading patterns in Drosophila melanogaster mitochondria based on their tRNA sequences: a unique wobble rule in animal mitochondria

    K Tomita, T Ueda, S Ishiwa, PF Crain, JA McCloskey, K Watanabe

    NUCLEIC ACIDS RESEARCH   27 ( 21 ) 4291 - 4297  1999年11月

     概要を見る

    Mitochondrial (mt) tRNA(Trp), tRNA(Ile), tRNA(Met), tRNA(Ser)GCU, tRNA(Asn) and tRNA(Lys) were purified from Drosophila melanogaster (fruit fly) and their nucleotide sequences were determined. tRNA(Lys) corresponding to both AAA and AAG lysine codons was found to contain the anticodon CUU, C34 at the wobble position being unmodified. tRNA(Met) corresponding to both AUA and AUG methionine codons was found to contain 5-formylcytidine (f(5)C) at the wobble position, although the extent of modification is partial. These results suggest that both C and f(5)C as the wobble bases at the anticodon first position (position 34) can recognize A at the codon third position (position 3) in the fruit fly mt translation system, tRNA(Ser)GCU Corresponding to AGU, AGC and AGA serine codons was found to contain unmodified G at the anticodon wobble position, suggesting the utilization of an unconventional G34-A3 base pair during translation. When these tRNA anticodon sequences are compared with those of other animal counterparts, it is concluded that either unmodified C or Gi at the wobble position can recognize A at the codon third position and that modification from A to t(6)A at position 37, 3'-adjacent to the anticodon, seems to be important for tRNA possessing C34 to recognize A3 in the mRNA in the fruit fly mt translation system.

    DOI PubMed CiNii

  • Gene contents and organization of a mitochondrial DNA segment of the squid Loligo bleekeri

    J Sasuga, S Yokobori, M Kaifu, T Ueda, K Nishikawa, K Watanabe

    JOURNAL OF MOLECULAR EVOLUTION   48 ( 6 ) 692 - 702  1999年06月

     概要を見る

    The nucleotide sequence of a 9240-base pair DNA fragment of the mitochondrial (mt) genome of a squid, Loligo bleekeri, was determined, in which 8 protein and 14 tRNA genes were identified. The gene organization of the mt-genome exhibits a greater resemblance to the gene organization of arthropods and a chiton, Katharina tunicata, than to those of a mussel, Mytilus edulis, and land snails. A cloverleaf-like structure was observed between the genes for subunits 4 and 5 of NADH dehydrogenase (ND4 and -5), which is considered to have originated from histidine tRNA, It is presumed that this structure functions as a transcriptional punctuation signal for the maturation of the ND4 and ND5 mRNAs.

    DOI PubMed CiNii

  • An extra tRNA(Gly)(U*CU) found in ascidian mitochondria responsible for decoding non-universal codons AGA AGG as glycine

    A Kondow, T Suzuki, S Yokobori, T Ueda, K Watanabe

    NUCLEIC ACIDS RESEARCH   27 ( 12 ) 2554 - 2559  1999年06月

     概要を見る

    Amino acid assignments of metazoan mitochondrial codons AGA/AGG are known to vary among animal species; arginine in Cnidaria, serine in invertebrates and stop in vertebrates. We recently found that in the mitochondria of the ascidian Halocynthia roretzi these codons are exceptionally used for glycine, and postulated that they are probably decoded by a tRNA(UCU), In order to verify this notion unambiguously, we determined the complete RNA sequence of the mitochondrial tRNA(UCU) presumed to decode codons AGA/AGG in the ascidian mitochondria, and found it to have an unidentified U derivative at the anticodon first position. We then identified the amino acids attached to the tRNA(U*CU), as well as to the conventional tRNA(Gly)(UCC) with an unmodified U34, in vivo. The results clearly demonstrated that glycine was attached to both tRNAs, Since no other tRNA capable of decoding codons AGA/AGG has been found in the mitochondrial genome, it is most probable that this tRNA(U*CU) does actually translate codons AGA/AGG as glycine in vivo. Sequencing of tRNA(Ser)(GCU), which is thought to recognize only codons AGU/AGC, revealed that it has an unmodified guanosine at position 34, as is the case with vertebrate mitochondrial tRNA(Ser)(GCU) for codons AGA/ AGG. It was thus concluded that in the ascidian, codons AGU/AGC are read as serine by tRNA(Ser)(GCU), whereas AGA/AGG are read as glycine by an extra tRNA(Gly)(U*CU). The possible origin of this unorthodox genetic code is discussed.

    DOI PubMed CiNii

  • Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation (vol 4, pg 257, 1998)

    Nitta, I, T Ueda, K Watanabe

    RNA-A PUBLICATION OF THE RNA SOCIETY   5 ( 5 ) 707 - 707  1999年05月

    その他  

  • The presence of pseudouridine in the anticodon alters the genetic code: a possible mechanism for assignment of the AAA lysine codon as asparagine in echinoderm mitochondria

    K Tomita, T Ueda, K Watanabe

    NUCLEIC ACIDS RESEARCH   27 ( 7 ) 1683 - 1689  1999年04月

     概要を見る

    It has been inferred from DNA sequence analyses that in echinoderm mitochondria not only the usual asparagine codons AAU and AAC, but also the usual lysine codon AAA, are translated as asparagine by a single mitochondrial (mt) tRNA(Asn) with the anticodon GUU. Nucleotide sequencing of starfish mt tRNA(Asn) revealed that the anticodon is G Psi U, U35 at the anticodon second position being modified to pseudouridine (Psi). In contrast, mt tRNA(Lys), corresponding to another lysine codon, AAG, has the anticodon CUU, mt tRNAs possessing anticodons closely related to that of tRNA(Asn), but responsible for decoding only two codons each-tRNA(His) tRNA(Asn) and tRNA(Tyr)-were found to possess unmodified U35 in all cases, suggesting the importance of Psi 35 for decoding the three codons. Therefore, the decoding capabilities of two synthetic Escherichia coli tRNA(Ala) variants with the anticodon G Psi U or GUU were examined using an E.coli in vitro translation system. Both tRNAs could translate not only AAC and AAU with similar efficiency, but also AAA with an efficiency that was similar to 2-fold higher in the case of tRNA(Ala)G Psi U than tRNA(Ala)GUU. These findings imply that Psi 35 of echinoderm mt tRNA(Asn) actually serves to decode the unusual asparagine codon AAA, resulting in the alteration of the genetic code in echinoderm mitochondria.

    DOI PubMed CiNii

  • A cytotoxic ribonuclease targeting specific transfer RNA anticodons

    T Ogawa, K Tomita, T Ueda, K Watanabe, T Uozumi, H Masaki

    SCIENCE   283 ( 5410 ) 2097 - 2100  1999年03月

     概要を見る

    The carboxyl-terminal domain of colicin E5 was shown to inhibit protein synthesis of Escherichia coli. Its target, as revealed through in vivo and in vitro experiments, was not ribosomes as in the case of E3, but the transfer RNAs (tRNAs) for Tyr, His, Asn, and Asp, which contain a modified base, queuine, at the wobble position of each anticodon. The E5 carboxyl-terminal domain hydrolyzed these tRNAs just on the 3' side of this nucleotide. Tight correlation was observed between the toxicity of E5 and the cleavage of intracellular tRNAs of this group, implying that these tRNAs are the primary targets of colicin E5.

    DOI PubMed CiNii

  • ウシミトコンドリア翻訳開始因子の機能解析

    土井啓利, 竹本千重, 小池智浩, 横川隆志, 竹内野乃, SPREMULLI L L, 上田卓也, 三浦謹一郎, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   22nd  1999年

    J-GLOBAL

  • ほ乳動物ミトコンドリアリボソームにおけるストークタンパク質の同定と機能解析

    寺崎真樹, 鈴木勉, 竹本千重, 花田孝雄, 和田明, 上田卓也, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   22nd  1999年

    J-GLOBAL

  • ヒトミトコンドリアペプチド鎖解離因子の機能解析

    新井芳実, 西内博章, 竹本千重, 鈴木勉, 上田卓也, 三浦謹一郎, 渡本公綱

    日本分子生物学会年会プログラム・講演要旨集   22nd  1999年

    J-GLOBAL

  • ミトコンドリアリボソームにおけるRNAからタンパク質への役割委譲

    鈴木勉, 寺崎真樹, 竹本千重, 花田孝雄, 和田明, 上田卓也, 渡辺公綱

    RNAミーティング   1st  1999年

    J-GLOBAL

  • Expression and characterization of bovine mitochondrial methionyl-tRNA transformylase

    N Takeuchi, T Ueda, K Watanabe

    JOURNAL OF BIOCHEMISTRY   124 ( 6 ) 1069 - 1071  1998年12月

     概要を見る

    Translational initiation in bacteria and some organelles such as mitochondria and chloroplasts requires formyl-methionyl-tRNA (fMet-tRNA), Methionyl-tRNA (Met-tRNA) undergoes formylation by methionyl-tRNA transformylase (MTF), and the resulting fMet-tRNA is utilized exclusively in the initiation process, The gene encoding mammalian mitochondrial MTF (MTFmt) was cloned recently. When the cDNA corresponding to mature MTFmt was cloned into an expression vector, no expression of MTFmt was observed. However, if the cDNA was fused with the histidine-tag sequence at the N-terminus, MTFmt could be expressed in Escherichia coli, The recombinant enzyme was purified by a single step on a histidine-binding metal a affinity column, We previously found that native MTFmt is able to formylate E. coli elongator Met-tRNA as well as the initiator Met-tRNA, The specific formylation of the initiator Met-tRNA by E. coli MTF is quite important in bacterial translational initiation, The purified recombinant MTFmt with the histidine-tag showed almost identical kinetic parameters to those of native MTFmt, This expression system is suitable for the rapid, efficient production of MTFmt in amounts adequate for further biophysical studies, which will provide another approach for elucidating the formylation mechanism, in addition to studies on E. coli MTF,

  • リボソームRNAの触媒機能

    新田 至, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   21   195 - 195  1998年12月

    CiNii

  • 牛ミトコンドアtRNA中に見いだされた未同定修飾塩基の構造解析

    鈴木 健夫, 鈴木 勉, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   21   441 - 441  1998年12月

    CiNii

  • LC/ESI MSを用いた修飾ウリジンの高感度, 特異的検出法の確立

    鈴木 勉, 鈴木 健夫, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   21   440 - 440  1998年12月

    CiNii

  • 多義語コドンの実験的検証

    鈴木 勉, 渡辺 公綱, 上田 卓也

    日本分子生物学会年会プログラム・講演要旨集   21   442 - 442  1998年12月

    CiNii

  • CCA末端修復酵素の反応機構の解析

    泊 幸秀, 鈴木 勉, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   21   442 - 442  1998年12月

    CiNii

  • 線形動物ミトコンドリアにおけるtRNAとEF-Tuの相互作用

    大槻 高史, 渡辺 洋一, 北 潔, 竹本 千重, 河合 剛太, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   21   442 - 442  1998年12月

    CiNii

  • 分節化構造を有するリボソームRNAの機能解析

    加藤 敬行, 新田 至, 上田 卓也, 渡辺 公鋼

    日本分子生物学会年会プログラム・講演要旨集   21   454 - 454  1998年12月

    CiNii

  • ウシミトコンドリア蛋白質合成系におけるセリルtRNA合成酵素のクローニング

    島田 信量, 横川 隆志, 竹内 野乃, 鈴木 勉, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   21   441 - 441  1998年12月

    CiNii

  • 翻訳に必須な因子の精製と再構成による生体外タンパク質合成系(Purified System)の構築

    井上 暁夫, 清水 義宏, 鈴木 勉, 横川 隆志, 西川 一八, 渡辺 公綱, 上田 卓也

    日本分子生物学会年会プログラム・講演要旨集   21   442 - 442  1998年12月

    CiNii

  • 乳児致死性心筋症患者由来点変異を持つミトコンドリアtRNA^<Ile>はアミノアシル化の効率が低い

    安川 武宏, 鈴木 勉, 上田 卓也, 太田 成男, 渡辺 公鋼

    日本分子生物学会年会プログラム・講演要旨集   21   455 - 455  1998年12月

    CiNii

  • Substrate recognition of tRNA (Guanosine-2 '-)-methyltransferase from Thermus thermophilus HB27

    H Hori, N Yamazaki, T Matsumoto, Y Watanabe, T Ueda, K Nishikawa, Kumagai, I, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   273 ( 40 ) 25721 - 25727  1998年10月

     概要を見る

    Transfer RNA (guanosine-2'-)-methyltransferase (Gm-methylase, EC 2.1.1.32) from Thermus thermophilus HB27 is one of the tRNA ribose modification enzymes. The broad substrate specificity of Gm-methylase has so far been elucidated using various species of tRNAs from native sources, suggesting that the common structures in tRNAs are recognized by the enzyme. In this study, by using 28 yeast tRNA(Phe) variants obtained by transcription with T7 RNA polymerase, it was revealed that the nucleotide residues G18 and G19 and the D-stem structure are essentially required for Gm-methylase recognition, and that the key sequence for the substrate is pyrimidine (Py)17G18G19. The other conserved sequences were found not to be essential, but U8, G15, G26, G46, U54, U55, and C56 considerably affected the methylation efficiency. These residues are located within a limited space embedded in the L-shaped three-dimensional structure of tRNA Therefore, disruption of the three-dimensional structure of the substrate tRNA is necessary for the catalytic center of Gm methylase to be able to access the target site in the tRNA, suggesting that the interaction of Gm-methylase with tRNA consists of multiple steps. This postulation was confirmed by inhibition experiments using nonsubstrate tRNA variants which functioned as competitive inhibitors against usual substrate tRNAs.

    DOI PubMed CiNii

  • Reconstitution of peptide bond formation with Escherichia coli 23S ribosomal RNA domains

    Nitta, I, Y Kamada, H Noda, T Ueda, K Watanabe

    SCIENCE   281 ( 5377 ) 666 - 669  1998年07月

     概要を見る

    It was recently demonstrated that peptide bond formation can occur using an Escherichia coli naked 23S ribosomal RNA without any of the ribosomal proteins. Here, the six domains of the 23S ribosomal RNA were individually synthesized and shown to be capable, when complexed together, of stimulating the reaction. Omission and addition experiments indicated that the activity could be reconstituted solely by domain V at a concentration 10 times higher than that of the intact 23S ribosomal RNA, whereas domain VI could enhance the activity in trans. These findings suggest that fragments of an RNA molecule have the ability to associate into a functional whole.

    DOI PubMed CiNii

  • A novel cell-free system for peptide synthesis driven by pyridine

    Nitta, I, H Nambu, T Okado, S Yoshinari, T Ueda, Y Endo, KH Nierhaus, K Watanabe

    BIOLOGICAL CHEMISTRY   379 ( 7 ) 819 - 829  1998年07月

     概要を見る

    Previously we demonstrated that ribosomes can synthesize polypeptides in the presence of high concentrations (40-60%) of pyridine without any protein factors, Here we analyze additional ribosomal parameters in 60% pyridine using Escherichia coli ribosomes, Ribosomal subunits once exposed to pyridine failed to re-associate to 70S ribosomes in aqueous buffer systems even in the presence of 20 mM Mg2+, whereas they formed 70S complexes in the presence of 60% pyridine, Two-dimensional gel electrophoresis of ribosomal proteins revealed that some proteins located at the protuberances of the large subunit, e, g. L7/L2 and L11 forming the elongation factor-binding domain, were released in the pyridine system. The aminoglycoside neomycin, a strong inhibitor of the ribosomal (factor-independent) translocation reaction, completely blocked poly(Phe) synthesis and translocation activities in the pyridine system, whereas these activities were not affected at all by gypsophilin, a ribotoxin that inhibits factor-dependent translocation, Another inhibitor of the ribosomal translocation, thiostrepton, had no effect concerning the two activites, which is consistent with the fact that this antibiotic requires L11 for its binding to the ribosome, These results suggest that the ribosomes can perform a translocation reaction in the pyridine system, but in a factor-independent (spontaneous) manner.

    DOI PubMed CiNii

  • Mammalian mitochondrial methionyl-tRNA transformylase from bovine liver - Purification, characterization, and gene structure

    N Takeuchi, M Kawakami, A Omori, T Ueda, LL Spremulli, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   273 ( 24 ) 15085 - 15090  1998年06月

     概要を見る

    The mammalian mitochondrial methionyl-tRNA transformylase (MTFmt) was partially purified 2,200-fold from bovine liver mitochondria using column chromatography, The polypeptide responsible for MTFmt activity was excised from a sodium dodecyl sulfate-polyacrylamide gel and the amino acid sequences of several peptides were determined. The cDNA encoding bovine MTFmt was obtained and its nucleotide sequence was determined. The deduced amino acid sequence of the mature form of MTFmt consists of 357 amino acid. residues. This sequence is about 30% identical to the corresponding Escherichia coli and yeast mitochondrial MTFs. Kinetic parameters governing the formylation of various tRNAs were obtained. Bovine MTFmt formylates its homologous mitochondrial methionyl-tRNA and the E. coli initiator methionyl-tRNA (Met-tRNA(fMet)) with essentially equal efficiency. The E. coli elongator methionyl-tRNA (Met-tRNA(mMet)) was also formylated although with somewhat less favorable kinetics. These results suggest that the substrate specificity of MTFmt is not as rigid as that of the E. coli MTF which clearly discriminates between the bacterial initiator and elongator Met-tRNAs. These observations are discussed in terms of the presence of a single tRNA(Met) gene in mammalian mitochondria.

    DOI

  • Possible involvement of Escherichia coli 23S ribosomal RNA in peptide bond formation

    Nitta, I, T Ueda, K Watanabe

    RNA-A PUBLICATION OF THE RNA SOCIETY   4 ( 3 ) 257 - 267  1998年03月

     概要を見る

    Experimental results are presented suggesting that 23S rRNA is directly involved in the peptide bond formation usually performed on the ribosome. Although several reports have indicated that the eubacterial peptidyltransferase reaction does not necessarily require all the ribosomal proteins, the reconstitution of peptidyltransferase activity by a naked 23S rRNA without the help of any of the ribosomal proteins has not been reported previously, It is demonstrated that an E. coli23S rRNA transcript synthesized by T7 RNA polymerase in vitro was able to promote peptide bond formation in the presence of 0.5% SDS. The reaction was inhibited by the peptidyltransferase-specific antibiotics chloramphenicol and carbomycin, and by digestion with RNases A and T1. Site-directed mutageneses at two highly conserved regions close to the peptidyltransferase center ring, G2252 to U2252 and C2507G2581 to U2507A2581, also suppressed peptide bond formation. These findings strongly suggest that 23S rRNA is the peptidyltransferase itself.

  • A novel wobble rule found in starfish mitochondria - Presence of 7-methylguanosine at the anticodon wobble position expands decoding capability of tRNA

    S Matsuyama, T Ueda, PF Crain, JA McCloskey, K Watanabe

    JOURNAL OF BIOLOGICAL CHEMISTRY   273 ( 6 ) 3363 - 3368  1998年02月

     概要を見る

    In the starfish mitochondrial (mt) genome, codons AGA and AGG (in addition to AGU and AGC) have been considered to be translated as serine. There is, however, only a single candidate mt tRNA gene responsible for translating these codons and it has a GCT anticodon sequence, but guanosine at the first position of the anticodon should base pair only with pyrimidines according to the conventional wobble rule. To solve this enigma, the mt tRNA(GCU)(ser) was purified, and sequence determination in combination with electrospray Liquid chromatography/mass spectrometry revealed that 7-methylguanosine is located at the first position of the anticodon. This is the first case in which a tRNA has been found to have 7-methylguanosine at the wobble position. It is suggested that methylation at N-7 of wobbling guanosine endows the tRNA with the capability of forming base pairs with all four nucleotides, A, U, G, and C, and expands the repertoire of codon-anticodon interaction. This finding indicates that a nonuniversal genetic code in starfish has been generated by base modification in the tRNA anticodon.

    DOI PubMed CiNii

  • Stability of mitochondrial tRNA molecules with pathogenic point mutations.

    Nucleic Acids. Symp. Ser.   39   257 - 258  1998年

  • 7-Methylguanosine at the anticodon wobble position of squid mitochondrial tRNA serGCU : molecular basis for assigment of AGA/AGG codons as serine in invertebrate mitochopndria.

    Biochim. Biophys. Acta   1399   78 - 82  1998年

    DOI

  • Ascidian mitochondrial tRNA(Met) possessing unique structural characteristics

    A Kondow, S Yokobori, T Ueda, K Watanabe

    NUCLEOSIDES & NUCLEOTIDES   17 ( 1-3 ) 531 - 539  1998年01月

     概要を見る

    Methionine tRNA was purified from muscle mitochondria of the ascidian Halocynthia roretzi and its RNA sequence was determined. Analysis of the nucleotide sequence revealed that unlike most metazoan mitochondrial tRNAs(Met), which have a highly conserved cytidine (C) or C-derivative at the wobble position, the H. roretzi mitochondrial tRNA(Met) possesses 5-carboxymethylaminomethyluridine (cmnm(5)U) at the first position of the anticodon. This is the first report of a single mitochondrial tRNA(Met) species having uridine (U) or a U-derivative at the wobble position.

  • The non-standard genetic code of Candida spp.: an evolving genetic code or a novel mechanism for adaptation?

    MAS Santos, T Ueda, K Watanabe, MF Tuite

    MOLECULAR MICROBIOLOGY   26 ( 3 ) 423 - 431  1997年11月

    書評論文,書評,文献紹介等  

     概要を見る

    A number of yeasts of the genus Candida translate the standard leucine-CUG codon as serine. This unique genetic code change is the only known alteration to the universal genetic code in cytoplasmic mRNAs, of either eukaryotes or prokaryotes, which involves reassignment of a sense codon. Translation of CUG as serine in these species is mediated by a novel serine-tRNA (ser-tRNA(CAG)), which uniquely has a guanosine at position 33, 5' to the anticodon, a position that is almost invariably occupied by a pyrimidine (uridine in general) in all other tRNAs. We propose that G-33 has two important functions: lowering the decoding efficiency of the ser-tRNA(CAG) and preventing binding of the leucyl-tRNA synthetase. This implicates this nucleotide as a key player in the evolutionary reassignment of the CUG codon. In addition, the novel ser-tRNA(CAG) has 1-methylguanosine (m(1)G-37) at position 37, 3' to the anticodon, which is characteristic of leucine, but not serine tRNAs. Remarkably, m(1)G-37 causes leucylation of the ser-tRNA(CAG) both in vitro and in vivo, making the CUG codon an ambiguous codon: the polysemous codon. This indicates that some Candida species tolerate ambiguous decoding and suggests either that (i) the genetic code change has not yet been fully established and is evolving at different rates in different Candida species; or (ii) CUG ambiguity is advantageous and represents the final stage of the reassignment. We propose that such dual specificity indicates that reassignment of the CUG codon evolved through a mechanism that required codon ambiguity and that ambiguous decoding evolved to generate genetic diversity and allow for rapid adaptation to environmental challenges.

    DOI PubMed CiNii

  • 多義語コドンの発見--複数のアミノ酸と結合するファジ-なtRNA

    上田 卓也, 鈴木 勉, 渡辺 公綱

    蛋白質核酸酵素   42 ( 11 ) 1815 - 1827  1997年08月

    CiNii

  • Assignment of imino proton signals of G-C base pairs and magnesium ion binding: An NMR study of bovine mitochondrial tRNA(GCU)(Ser) lacking the entire D arm

    Hayashi, I, T Yokogawa, G Kawai, T Ueda, K Nishikawa, K Watanabe

    JOURNAL OF BIOCHEMISTRY   121 ( 6 ) 1115 - 1122  1997年06月

     概要を見る

    The mammalian mitochondrial tRNA(GCU)(Ser) (mt tRNA(GCU)(Ser)) has a unique structure in that it lacks the whole D arm, To elucidate its higher-order structure, we synthesized unmodified bovine mt tRNA(GCU)(Ser) using T7 RNA polymerase and measured its H-1-NMR spectrum in the imino proton region, Although the imino proton signals heavily overlapped, we succeeded in assigning all the seven imino proton signals of the G-C base pairs by a combination of base replacement and N-15-labeling of the G residues of a whole tRNA molecule or of the 3'-half fragment, The results indicate that the tRNA possesses the secondary structure that has been supposed on the basis of biochemical studies, Analysis of the effect of the magnesium concentration on the G-C pairs suggests that the acceptor and T stems do not form a co-axial helix, and that the core region of the tRNA does not interact with magnesium ions, These features are significantly different from those of canonical tRNAs, Despite this, it is very likely that the tRNA as a whole takes a nearly L-shape tertiary structure.

  • The 'polysemous' codon - A codon with multiple amino acid assignment caused by dual specificity of tRNA identity

    T Suzuki, T Ueda, K Watanabe

    EMBO JOURNAL   16 ( 5 ) 1122 - 1134  1997年03月

     概要を見る

    In some Candida species, the universal CUG leucine codon is translated as serine. However, in most cases, the serine tRNAs responsible for this non-universal decoding (tRNA(Ser)CAG) accept in vitro not only serine, but also, to some extent, leucine. Nucleotide replacement experiments indicated that m(1)G37 is critical for leucylation activity. This finding was supported by the fact that the tRNA(Ser)CAGs possessing the leucylation activity always have m(1)G37, whereas that of Candida cylindracea, which possesses no leucylation activity, has A37. Quantification of defined aminoacetylated tRNAs in cells demonstrated that 3% of the tRNA(Ser)CAGs possessing m(1)G37 were, in fact, charged with leucine in vivo. A genetic approach using an auxotroph mutant of C.maltosa possessing this type of tRNA(Ser)CAG also suggested that the URA3 gene inactivated due to the translation of CUG as serine was rescued by a slight incorporation of leucine into the polypeptide, which demonstrated that the tRNA charged with multiple amino acids could participate in the translation. These findings provide the first evidence that two distinct amino acids are assigned by a single codon, which occurs naturally in the translation process of certain Candida species. We term this novel type of codon a 'polysemous codon'.

    DOI PubMed CiNii

  • Evolution of pulmonate gastropod mitochondrial genomes: Comparisons of gene organizations of Euhadra, Cepaea and Albinaria and implications of unusual tRNA secondary structures

    N Yamazaki, R Ueshima, JA Terrett, S Yokobori, M Kaifu, R Segawa, T Kobayashi, K Numachi, T Ueda, K Nishikawa, K Watanabe, RH Thomas

    GENETICS   145 ( 3 ) 749 - 758  1997年03月

     概要を見る

    Complete gene organizations of the mitochondrial genomes of three pulmonate gastropods, Euhadra herklotsi, Cepaea nemoralis and Albinaria coerulea, permit comparisons of their gene organizations. Euhadra and Cepaea are classified in the same superfamily, Helicoidea, yet they show several differences in the order of tRNA and protein coding genes. Albinaria is distantly related to the other two genera but shares the same gene order in one part of its mitochondrial genome with Euhadra and in another part with Cepaea. Despite their small size (14.1-14.5 kbp), these snail mtDNAs encode 13 protein genes, two rRNA genes and at least 22 tRNA genes. These genomes exhibit several unusual or unique features compared to other published metazoan mitochondrial genomes, including those of other molluscs. Several tRNAs predicted from the DNA. sequences possess bizarre structures lacking either the T stem or the D stem, similar to the situation seen in nematode mt-tRNAs. The acceptor stems of many tRNAs show a considerable number of mismatched basepairs, indicating that the RNA editing process recently demonstrated in Euhadra is widespread in the pulmonate gastropods. Strong selection acting on mitochondrial genomes of these animals would have resulted in frequent occurrence of the mismatched basepairs in regions of overlapping genes.

  • Primary sequence of mitochondrial tRNA(Arg) of a nematode Ascaris suum: Occurrence of unmodified adenosine at the first position of the anticodon

    Y Watanabe, H Tsurui, T Ueda, R FurusihimaShimogawara, S Takamiya, K Kita, K Nishikawa, K Watanabe

    BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION   1350 ( 2 ) 119 - 122  1997年02月

     概要を見る

    Mitochondrial tRNA(Arg) from a nematode, Ascaris suum, was purified and sequenced at the RNA level. An unmodified adenosine was found to exist at the anticodon first position, suggesting that, contrary to the conventional wobble rule, the anticodon ACG of the tRNA can translate all the CGN codons.

    DOI

  • "cDNA sequence of a translational elongation factor Ts homologue from Caenorhabditis elegans : mitochondrial factor-specific features found in the nematode homologue peptides.

    Biochim. Biophys. Acta   1353   7 - 12  1997年

    DOI

  • Two nucleotides 5'-adijacent to the anticodon of rat cytoplasmic tRNA Asp are not edited ?

    Biochimie   78   1001 - 1006  1997年

    DOI

  • ウシミトコンドリア解離因子のクローニング

    西内博章, 竹本千重, 上田卓也, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   20th  1997年

    J-GLOBAL

  • RNA editing in the acceptor stem of squid mitochondrial tRNA(Tyr)

    K Tomita, T Ueda, K Watanabe

    NUCLEIC ACIDS RESEARCH   24 ( 24 ) 4987 - 4991  1996年12月

     概要を見る

    In squid (Loligo bleekeri) mitochondria, the two 3'-terminal nucleotides (G72-G73) of the tRNA(Tyr) gene overlap with the two 5'-terminal nucleotides (G1-G2) of the downstream tRNA(Cys) gene, To elucidate the processing mechanism(s) of the tRNA molecules derived from this region, tRNAs were analyzed by sequencing cDNAs synthesized from circularized tRNAs, Nucleotides G1-G2 in tRNA(Cys) appeared to be without post-transcriptional conversion, whereas CCA was post-transcriptionally added to the 3'-terminus. In contrast, in the majority of tRNAs(Tyr), G72-G73 were found to be converted to A72-A73, accompanied by the CCA addition, These results indicate that a precursor of tRNA(Tyr) is processed at U71 and two adenosines are attached prior to the CCA addition. Thus, we suggest that 5' processing of the precursor tRNA dominates 3' processing and maturation of the tRNA is mediated by a polyadenylylation enzyme in the mitochondria, a scenario which is consistent with the editing process proposed in land snail mitochondria, We also obtained intermediates, such as a premature tRNA lacking CCA that terminated at U71 and one with a single adenosine attached at position 72, which support the suggested maturation process, However, although we failed to detect a tRNA(Cys) lacking G1-G2 at the 5'-terminus, we obtained cDNAs for tRNA(Tyr) with G72-G73 and the CCA terminus. This inconsistent result suggests the co-existence of another process(es) in the maturation of these tRNA molecules in squid mitochondria.

    DOI PubMed CiNii

  • 蛋白質合成系におけるリボソームRNAの機能

    新田 至, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   19   35 - 35  1996年08月

    CiNii

  • ヌクレアーゼ型コリシンE4とE5の作用機構の研究

    郡司 義哉, 小川 哲弘, 新田 至, 上田 卓也, 渡辺 公綱, 正木 春彦, 魚住 武司

    日本分子生物学会年会プログラム・講演要旨集   19   467 - 467  1996年08月

    CiNii

  • ウシミトコンドリアの翻訳開始機構におけるMet-tRNA transformylase(MTF)

    竹内 野乃, 川上 実, 小池 智浩, 竹本 千重, 横川 隆志, 上田 卓也, SPREMULLI L. L., 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   19   463 - 463  1996年08月

    CiNii

  • 線形動物EF-Tu相同蛋白質の大腸菌における発現

    渡邊 洋一, 北 潔, 上代 淑人, 上田 卓也, 渡辺 公綱

    日本分子生物学会年会プログラム・講演要旨集   19   464 - 464  1996年08月

    CiNii

  • Pyridine-promoted factor- and energy-free peptide synthesis systems prepared from various organisms including prokaryote, eukaryote, and mitochondria

    T Nojima, Nitta, I, T Ueda, K Watanabe

    JOURNAL OF BIOCHEMISTRY   119 ( 6 ) 1076 - 1079  1996年06月

     概要を見る

    We demonstrate here that ribosomes from not only Escherichia coli and Thermus thermophilus [Nitta ef al. (1994) al J. Biochem. 115, 803-807; ibid., (1995) 118, 841-849] but also yeast and bovine mitochondria catalyze peptide synthesis promoted by a high concentration of pyridine in the absence of soluble protein factors and chemical energy sources, and compare some characteristic features of the reactions among these organisms. Sensitivities against antibiotics, chloramphenicol and cycloheximide, showed the same tendency to those in the in vitro aqueous translation systems of these organisms, suggesting that the basic mechanism for peptide synthesis is the same among these organisms. The optimal concentration of pyridine was centered at 50% for all systems, although the dependencies on the pyridine concentrations and the yields of the products were different from one another. All these systems required Mg2+, and only mitochondrial system showed the extra Mn2+-requirement, which enhanced the yield by several fold. The optimum reaction temperatures coincided closely with the growing temperatures of the organisms except for the mitochondrial system, which showed the highest activity above 80 degrees C, The rationale far these observations remains to be solved.

  • コリシンE4とE5の持つヌクレアーゼ活性に関する研究 : 微生物

    小川 哲弘, 郡司 義哉, 新田 至, 上田 卓也, 渡辺 公綱, 正木 春彦, 魚住 武司

    日本農藝化學會誌   70   320 - 320  1996年03月

    CiNii

  • A new method for identifying the amino acid attached to a particular RNA in the cell

    Tsutomu Suzuki, Takuya Ueda, Kimitsuna Watanabe

    FEBS Letters   381 ( 3 ) 195 - 198  1996年03月

     概要を見る

    To investigate the function of tRNAs or any other aminoacylable RNAs in vivo, it is important to be able to estimate the amounts and species of aminoacylated RNAs in living cells. We have developed a method of analyzing amino acids attached to particular tRNAs obtained from cells. After the ester bond between the amino acid and the 3'-adenosine moiety of a specific aminoacyl-tRNA is stabilized by acetylation of the amino acid with [14C]acetic anhydride, the aminoacyl-tRNA can be fished out with a solid-phase-attached DNA probe. The 14C-labeled acetylamino acid is then released from the thus purified acetyl-aminoacyl-tRNAs by alkaline treatment and detected by TLC analysis.

    DOI PubMed CiNii

  • Structural feature of the initiator tRNA gene from Pyrodictium occultum and the thermal stability of its gene product, tRNA(i)(Met)

    C Ushida, T Muramatsu, H Mizushima, T Ueda, K Watanabe, KO Stetter, PF Crain, JA McCloskey, Y Kuchino

    BIOCHIMIE   78 ( 10 ) 847 - 855  1996年

     概要を見る

    Pyrodictium occultum is a hyperthermophilic archaeum that grows optimally at 105 degrees C. To study how tRNA molecules in P occultum are thermally stabilized, we isolated the initiator tRNA gene from the organism using a synthetic DNA probe of 74 bp containing the known nucleotide sequences that are conserved in archaeal initiator tRNAs. A HindIII fragment of 700 bp containing the Pyrodictium initiator tRNA gene was cloned and sequenced by cycle sequencing. The nucleotide sequence revealed that the Pyrodictium initiator tRNA gene has no introns, and that the 3' CCA terminus is encoded. The tRNA gene also contained a unique TATA-like sequence, AAGCTTATAA, which is likely the promoter proposed for archaeal tRNA genes, -50 bp upstream of the 5' end of the tRNA coding region. In the region adjacent to the 3' end of the tRNA coding region, there was a six G-C base pair inverted repeat followed by a C-rich sequence like the p-independent transcription termination signal of bacterial genes. The Pyrodictium initiator tRNA sequence predicted from the gene sequence contained all of the nucleotide residues A1, A37, U54, A57, U60, and U72, in addition to three G-C base pairs in the anticodon stem region, which are characteristic of archaeal initiator tRNAs. The melting temperature (T-m) of the unmodified initiator tRNA synthesized in vitro using the cloned tRNA gene as a template was 80 degrees C, which is only two degrees lower than that calculated from the G-C content in the stem regions of the tRNA. In contrast, the T-m of the natural initiator tRNA isolated from P occultum was over 100 degrees C. Analysis of digests of purified Pyrodictium initiator tRNA by means of HPLC-mass spectrometry and [P-32] post-labeling, indicated that the tRNA contains a variety of modified nucleosides. These results suggest that the extraordinarily high melting temperature of P occultum tRNA(i)(Met) is due to posttranscriptional modification.

    DOI PubMed

  • ピリジンにより活性化された種々の生体外蛋白質合成系

    野島高彦, 新田至, 竹本千重, 上田卓也, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   18th   198  1995年11月

    J-GLOBAL

  • FACTOR-FREE AND ENERGY-FREE PEPTIDE-SYNTHESIS PROMOTED BY AROMATIC TERTIARY-AMINES INCLUDING NUCLEIC ACID-RELATED COMPOUNDS

    NITTA, I, T UEDA, K WATANABE

    JOURNAL OF BIOCHEMISTRY   118 ( 4 ) 850 - 854  1995年10月

     概要を見る

    We have already reported a novel, in vitro translation system, promoted by pyridine instead of the usual protein factors and energy sources, which consists of only salt-washed ribosomes from Escherichia coli, aminoacyl-tRNA, a template RNA, Na+ and Mg2+ cations, and 40-60% pyridine [Nitta et al. (1994) J. Biochem, 115, 803-807 and the accompanying paper]. Here we show that in this system, pyridine can be replaced not only by pyridine derivatives but also by nucleobases or nucleosides, demonstrating that any compound harboring an aromatic tertiary amine within the molecule possesses such promoting activity. These compounds may serve to assist the peptide bond formation catalyzed by peptidyltransferase within ribosomes. The finding that nucleobases and nucleosides can play such a role in this reaction implies the possibility that these compounds were directly involved in the premordial translation system.

  • TEMPLATE-DEPENDENT POLYPEPTIDE-SYNTHESIS IN A FACTOR-FREE AND ENERGY-FREE TRANSLATION SYSTEM PROMOTED BY PYRIDINE

    NITTA, I, T UEDA, T NOJIMA, K WATANABE

    JOURNAL OF BIOCHEMISTRY   118 ( 4 ) 841 - 849  1995年10月

     概要を見る

    We demonstrate here that a high concentration (40-70%) of pyridine, an aromatic tertiary amine catalyst, is able to promote translation on ribosomes without the presence of soluble protein factors or chemical energy sources, Compared with Monro's fragment reaction [Methods Enzymol. 20, 472-481 (1971)], which reflects only the peptidyltransferase step, this novel translation system can produce polypeptides with chain lengths of at least several tens of residues depending on the template RNA. In the presence of 60% pyridine, poly(U) and poly(UC) promoted incorporation of the respective amino acids, phenylalanine and serine-leucine, twofold, whereas poly(A) promoted the incorporation of lysine by only 25%. The degrees of polymerization of phenylalanine and lysine were up to the decamer and around 40mer, respectively, In poly(UC)-dependent oligo(serine-leucine) synthesis, oligopeptides with a serine and leucine alternate sequence were the main products, This novel pyridine system evidently differs from the non-enzymatic translation system reported by Gavrilova and Spirin [FEBS Lett. 17, 324-326 (1971)]; the former system displays partial resistance toward deproteinization reagents such as SDS and proteinase K, whereas the latter system is completely sensitive.

  • 芳香族三級アミンを用いた新規な生体外タンパク質合成系とその翻訳系の起源研究への意義

    新田 至, 上田 卓也, 野島 高彦, 渡辺 公綱

    Viva origino   23 ( 4 ) 209 - 221  1995年

    CiNii

  • THE ABILITY OF BOVINE MITOCHONDRIAL TRANSFER RNA(MET) TO DECODE AUG AND AUA CODONS

    C TAKEMOTO, T KOIKE, T YOKOGAWA, L BENKOWSKI, LL SPREMULLI, TA UEDA, K NISHIKAWA, K WATANABE

    BIOCHIMIE   77 ( 1-2 ) 104 - 108  1995年

     概要を見る

    The ability of bovine mitochondrial tRNA(Met) with the anticodon f(5)CAU (where f(5)C is 5-formylcytidine) to decode AUG and AUA codons was examined in a codon-dependent ribosomal binding assay. The AUG codon stimulated the binding of Met tRNA(Met) to mitochondrial ribosomes in the presence of EF-Tu/Ts(mt). In contrast, the AUA codon did not promote the binding to mitochondrial Met-tRNA to the ribosome. To investigate the translation of the AUG and AUA codons more fully, an in vitro, translation system from bovine liver mitochondria was developed. The activity of this system was greatly enhanced by the addition of 1 mM spermine and reached about half the activity observed with a comparable translational system from E coli. Two types of mRNA containing either AUG or AUA codons were synthesized using T7 RNA polymerase to transcribe their chemically synthesized genes. In the E coli system, the AUG-containing mRNA was translated as Met and the AUA-containing mRNA was translated as Ile. The AUG-containing mRNA but not the AUA-containing mRNA was translated as Met by the mitochondrial translational system. The process by which the AUA codon is translated as Met in the mitochondrial system remains to be clarified.

    DOI

  • ミトコンドリアにおけるtRNAMetの変則暗号認識機構

    竹本千重, 大槻高史, 上田卓也, 河合剛太, 高久洋, SPREMULLI L L

    日本分子生物学会年会プログラム・講演要旨集   18th  1995年

    J-GLOBAL

  • A UGU SEQUENCE IN THE ANTICODON LOOP IS A MINIMUM REQUIREMENT FOR RECOGNITION BY ESCHERICHIA-COLI TRANSFER-RNA-GUANINE TRANSGLYCOSYLASE

    S NAKANISHI, T UEDA, H HORI, N YAMAZAKI, N OKADA, K WATANABE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 51 ) 32221 - 32225  1994年12月

     概要を見る

    Escherichia coli tRNA-guanine transglycosylase is an enzyme which catalyzes replacement of guanine (G(34)) Of tRNA(Asp), tRNA(Asn), tRNA(His) and tRNA(Tyr) by free guanine or free preQ(1) base by a base exchange reaction in the biosynthesis of queuosine (Q) (Okada, N., and Nishimura, S. (1979) J. Biol. Chem. 254, 3061-3066). The gene encoding for this enzyme was amplified from the E. coli genome by polymerase chain reaction and inserted into an overexpression vector, pJLA503, The enzyme was overexpressed by heat induction in E. coli transformed by this recombinant plasmid and purified to homogeneity by two column chromatographies. The sequence requirement in tRNA for recognition by this enzyme was investigated using minihelices corresponding to the anticodon arm of E. coli tRNA(His). Two uridine residues (U-33, U-35) were found to be prerequisite for such recognition by this enzyme, Position 32 required pyrimidines, because the enzyme activity toward the minihelices was markedly reduced or entirely lost when this residue was replaced by purines or was deleted, Adenosine at position 37 and the G(30)-C-40 base pair were not essential despite their conservation. Our results suggest that the enzyme recognizes the U-33-G(34)-U-35 sequence in the anticodon loop and not the tertiary structure of tRNA itself.

  • HIGHER-ORDER STRUCTURE OF BOVINE MITOCHONDRIAL TRNA(SER)UGA - CHEMICAL MODIFICATION AND COMPUTER MODELING

    Y WATANABE, G KAWAI, T YOKOGAWA, N HAYASHI, Y KUMAZAWA, T UEDA, K NISHIKAWA, HIRAO, I, K MIURA, K WATANABE

    NUCLEIC ACIDS RESEARCH   22 ( 24 ) 5378 - 5384  1994年12月

     概要を見る

    On the basis of enzymatic probing and phylogenetic comparison, we have previously proposed that mammalian mitochondrial tRNA(Ser) (anticodon UGA) possess a slightly altered cloverleaf structure in which only one nucleotide exists between the acceptor stem and D stem (usually two nucleotides) and the anticodon stem consists of six base pairs (usually five base pairs) [Yokogawa at al. (1991) Nucleic Acids Res. 19, 6101- 6105]. To ascertain whether such tRNAs(Ser) can be folded into a normal L-shaped tertiary structure, the higher-order structure of bovine mitochondrial tRNA(Ser)UGA was examined by chemical probing using dimethylsulfate and diethylpyrocarbonate, and on the basis of the results a tertiary structure model was obtained by computer modeling. It was found that a one-base-pair elongation in the anticodon stem was compensated for by multiple-base deletions in the D and extra loop regions of the tRNA(Ser)UGA, which resulted in preservation of an L-shaped tertiary structure similar to that of conventional tRNAs. By summarizing the findings, the general structural requirements of mitochondrial tRNAs necessary for their functioning in the mitochondrial translation system are considered.

    DOI PubMed CiNii

  • ショウジョウバエミトコンドリアにおけるコドン認識機構

    富田耕造, 上田卓也, 横堀伸一, 石和貞男, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   17th   286  1994年11月

    J-GLOBAL

  • キョク皮動物ミトコンドリアに見られる特殊なコドン‐アンチコドン対合関係

    荒木武義, 大久保頼聡, 横堀伸一, 渡辺洋一, 上田卓也, 西川一八, 三浦謹一郎, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   17th   286  1994年11月

    J-GLOBAL

  • AGNコドンを認識する動物ミトコンドリアtRNA

    近藤晶子, 竹本千重, 横堀伸一, 横川隆志, 上田卓也, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   17th   286  1994年11月

    J-GLOBAL

  • PRIMARY-ORDER AND HIGHER-ORDER STRUCTURES OF NEMATODE (ASCARIS-SUUM) MITOCHONDRIAL TRANSFER-RNAS LACKING EITHER THE T-STEM OR D-STEM

    YI WATANABE, H TSURUI, T UEDA, R FURUSHIMA, S TAKAMIYA, K KITA, K NISHIKAWA, K WATANABE

    JOURNAL OF BIOLOGICAL CHEMISTRY   269 ( 36 ) 22902 - 22906  1994年09月

     概要を見る

    By fractionation using polyacrylamide gel electrophoresis and/or a preparative hybrid selection method employing solid-phase DNA probes, we prepared and characterized mitochondrial tRNAs from the body wall muscle of Ascaris suum, all of which are thought to lack either the T stem or the D stem from their gene sequences (Okimoto, R., and Wolstenholme, D. R. (1990) EMBO J. 10, 3405-3411). Some of the partially purified tRNAs were appreciably aminoacylated with an extract of A. suum mitochondria. The three species sequenced had CCA sequence at their 3'-ends, and tRNA(Met) had 5-formylcytidine at the anticodon first position, a new modified nucleoside found at the same position of bovine mitochondrial tRNA(Met) (Moriya, J., Yokogawa, T., Wakita, K., Ueda, T., Nishikawa, K., Crain, P. F., Hashizume, T., Pomerantz, S. C., McCloskey, J. A., Kawai, G., Hayashi, N., Yokoyama, S., and Watanabe, K. (1994) Biochemistry 33, 2234-2239). Enzymatic probing of these tRNAs supported the secondary structural model proposed by Okimoto and Wolstenholme in the reference cited above. Chemical probing of tRNA(Phe) demonstrated the existence of tertiary interactions between the (T arm-variable loop)-replacement loop and the D arm. The results suggest that these tertiary interactions enable the bizarre tRNAs of nematode mitochondria to maintain an L-shape-libe structure in order to function in the nematode mitochondrial translation system.

  • RELATIONSHIP AMONG COELACANTHS, LUNGFISHES, AND TETRAPODS - A PHYLOGENETIC ANALYSIS BASED ON MITOCHONDRIAL CYTOCHROME-OXIDASE-I GENE-SEQUENCES

    S YOKOBORI, M HASEGAWA, T UEDA, N OKADA, K NISHIKAWA, K WATANABE

    JOURNAL OF MOLECULAR EVOLUTION   38 ( 6 ) 602 - 609  1994年06月

     概要を見る

    To clarify the relationship among coelacanths, lungfishes, and tetrapods, the amine acid sequences deduced from the nucleotide sequences of mitochondrial cytochrome oxidase subunit I(COI) genes were compared. The phylogenetic tree of these animals, including the coelacanth Latimeria chalumnae and the lungfish Lepidosiren paradoxa, was inferred by several methods. These analyses consistently indicate a coelacanth/lungfish clade, to which little attention has been paid by previous authors with the exception of some morphologists. Overall evidence of other mitochondrial genes reported previously and the results of this study equally support the coelacanth/lungfish and lungfish/tetrapod clades, ruling out the coelacanth/tetrapod clade.

  • NUCLEASE RESISTANCE OF AN EXTRAORDINARILY THERMOSTABLE MINI-HAIRPIN DNA FRAGMENT, D(GCGAAGC) AND ITS APPLICATION TO IN-VITRO PROTEIN-SYNTHESIS

    S YOSHIZAWA, T UEDA, Y ISHIDO, K MIURA, K WATANABE, HIRAO, I

    NUCLEIC ACIDS RESEARCH   22 ( 12 ) 2217 - 2221  1994年06月

     概要を見る

    The nuclease resistance of a short, thermostable mini-hairpin, d(GCGAAGC), and other related hairpins was examined. Hairpins possessing a purine-rich (GAA) or (GAAA) loop appeared to be more resistant against nucleases than those with a pyrimidine-rich loop or single-stranded oligomers. Among 8 kinds of oligodeoxyribonucleotides examined, the fragment most resistant against nucleases was a hairpin with the sequence of d(CGCGAAGCG). This hairpin was then utilized for the stabilization of mRNA in an in vitro translation system; the 3'-terminal region of an mRNA was hybridized with an oligodeoxyribonucleotide including the sequence complementary to the 3'-terminus of the mRNA tagged with the nuclease-resistant d(CGCGAAGCG) hairpin sequence. By using this method, dihydrofolate reductase (DHFR) mRNA was stabilized against nucleases contaminating a cell-free translation system of E.coli, with a consequent increase in protein synthesis efficiency of 200%.

    DOI PubMed CiNii

  • SUBSTRATE-SPECIFICITY OF TRANSFER-RNA (ADENINE-1-)-METHYLTRANSFERASE FROM THERMUS-THERMOPHILUS HB27

    N YAMAZAKI, H HORI, K OZAWA, S NAKANISHI, T UEDA, KUMAGAI, I, K WATANABE, K NISHIKAWA

    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY   58 ( 6 ) 1128 - 1133  1994年06月

     概要を見る

    tRNA (adenine-1-)-methyltransferase was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB27, by several steps of column chromatographies. The molecular weight of this enzyme was about 60,000 as analyzed by SDS polyacrylamide gel electrophoresis. K-m for E. coli tRNA(2)(Glu) was 100 nM and that for the methyl group donor, S-adenosyl-L-methionine, was 7.8 mu M. The substrate specificity of the enzyme was investigated by using T7 RNA polymerase transcripts and tRNA fragments obtained by partial digestion with RNases. The enzyme was able to transfer the methyl group to the 3'-half fragment of E. coli initiator tRNA, however, the extent of methylation was elevated by more than five times when the 5'-half fragment was added and annealed to the 3'-half. This indicates that the main recognition site of the enzyme is within the 3'-half region of tRNA molecule, while the tertiary interaction between the T-loop and the D-loop is very effective for the adequate methylation reaction.

    DOI PubMed

  • EFFICIENT EXPRESSION OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE GENE BY AN IN-VITRO TRANSLATION SYSTEM USING PHOSPHOROTHIOATE MESSENGER-RNA

    H TOHDA, N CHIKAZUMI, T UEDA, K NISHIKAWA, K WATANABE

    JOURNAL OF BIOTECHNOLOGY   34 ( 1 ) 61 - 69  1994年04月

     概要を見る

    Dihydrofolate reductase (DHFR) of Escherichia call (E. coli) was synthesized in a cell-free translation system of E. coli directed by phosphorothioate-containing mRNA (thio-mRNA) which was polymerized by an in vitro transcription of the DHFR gene in the presence of S-P diastereomers of ribonucleoside 5'-O-(1-thiotriphosphates). The molecular weights of the products thus obtained were identical to those with the unsubstituted mRNA. Furthermore, the thio-mRNA for DHFR showed higher translational activities than the corresponding unsubstituted mRNA. It is suggested that this effectiveness resulted from the higher stability of thio-mRNA in the cell-free translation system. Amongst the various types of thio-mRNAs, the single substitution of adenosine residues was most effective in translational activity. This higher translational activity of thio-mRNA compared with the unsubstituted mRNA was also demonstrated in a continuous flow cell-free system originally developed by Spirin et al. (1988). Therefore, introduction of sulfur atoms into phosphodiester bonds of mRNA appears to be a useful strategy for the stabilization of mRNA in large-scale protein production in vitro.

    DOI

  • TEMPLATE-DEPENDENT PEPTIDE FORMATION ON RIBOSOMES CATALYZED BY PYRIDINE

    NITTA, I, T UEDA, K WATANABE

    JOURNAL OF BIOCHEMISTRY   115 ( 4 ) 803 - 807  1994年04月

     概要を見る

    We found that poly(U)-dependent polyphenylalanine synthesis on Escherichia coli ribosomes was extremely accelerated in the presence of a high concentration of pyridine. In this reaction, chemical energy sources, such as ATP and G;TP, and soluble protein factors are not required, but the template and ribosomes are essential for the progress of the reaction. The reaction was inhibited by the antibiotics which inhibit the usual bacterial translation process on the ribosomes. These observations clearly demonstrate that the pyridine-catalyzed amino acid condensation reaction proceeds on the ribosome.

  • A NOVEL MODIFIED NUCLEOSIDE FOUND AT THE FIRST POSITION OF THE ANTICODON OF METHIONINE TRANSFER-RNA FROM BOVINE LIVER-MITOCHONDRIA

    J MORIYA, T YOKOGAWA, K WAKITA, T UEDA, K NISHIKAWA, PF CRAIN, T HASHIZUME, SC POMERANTZ, JA MCCLOSKEY, G KAWAI, N HAYASHI, S YOKOYAMA, K WATANABE

    BIOCHEMISTRY   33 ( 8 ) 2234 - 2239  1994年03月

     概要を見る

    Methionine tRNA was purified from bovine liver mitochondria, and its nucleotide sequence was determined. The tRNA possesses only three posttranscriptionally modified nucleosides, two pseudouridines in the anticodon and T stems and a previously unknown nucleoside specified by the gene sequence as cytidine, in the first position of the anticodon. Structure analysis of the anticodon nucleoside by mass spectrometry revealed a molecular mass 28 Da greater than that of cytidine, and unmodified ribose, with substitution at C-5 implied by hydrogen-deuterium exchange experiments. Proton NMR of the intact tRNA showed presence of a formyl moiety, thus leading to the candidate structure 5-formylcytidine (f(5)C), not a previously known compound. The structure assignment was confirmed by chemical synthesis and comparison of data from combined HPLC/mass spectrometry and proton NMR for the natural and synthetic nucleosides. The potential function of f(5)C in the tRNA(Met) anticodon is discussed with regard to codon-anticodon interactions.

    DOI

  • HIGHER-ORDER STRUCTURE OF BOVINE MITOCHONDRIAL TRNA(PHE) LACKING THE CONSERVED GG AND T-PSI-CG SEQUENCES AS INFERRED BY ENZYMATIC AND CHEMICAL PROBING

    K WAKITA, Y WATANABE, T YOKOGAWA, Y KUMAZAWA, S NAKAMURA, T UEDA, K WATANABE, K NISHIKAWA

    NUCLEIC ACIDS RESEARCH   22 ( 3 ) 347 - 353  1994年02月

     概要を見る

    Bovine mitochondrial (mt) phenylalanine tRNA (tRNA(Phe)), which lacks the 'conserved' GG and T Psi YCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and it higher-order structure was investigated using RNase T-2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNase T-2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected form the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNA(Phe) genes from various organisms, it was inferred that bovine mt tRNA(Phe) lacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNA(Phe) to preserve its L-shaped higher-order structure.

    DOI PubMed CiNii

  • Characterization of serine and leucine tRNAs in an asporogenic yeast Candida cylindraceaand evolutionary implications of genes for tRNASerCAG responsible for translation of a non-universal genetic code

    Tsutomu Suzuki, Takuya Ueda, Takashi Yokogawa, Kazuya Nishikawa, Kimitsuna Watanabe

    Nucleic Acids Research   22 ( 2 ) 115 - 123  1994年01月

     概要を見る

    Five serine and three leucine isoacceptor tRNAs were purified from the asporogenic yeast Candida cylindracea, in which codon CUG is translated as serine Instead of leucine [1], and their primary structures were determined. From the wobble hypothesis [2], it was assumed that one of the tRNALeu species (Leu1), with the antlcodon CmAA, corresponded to the UUG leucine codon, and that the remaining two leucine tRNAs (Leu2 and Leu3), with the same IAG antlcodon sequence, would decode the CUU, CUC and CUA codons as leucine, but not the CUG codon
    this was clarified by an In vitro translation experiment with C.cylindracea using synthetic mRNAs containing the CUA or CUG codons. One of the serine tRNAs (Ser1) has already been demonstrated to have the antlcodon CAG and to be responsible for translation of the codon CUG In C.cylindracea [3]. Three of the other species of tRNASer(Ser2,3 and 4), with the anticodon sequences cm5UGA, IGA and CGA, can translate all four codons in the UCN codon box, while the remaining species (Ser5), with the anticodon GCU, corresponds to AGU and AGC serine codons. The gene sequences for these five serine and three leucine tRNAs were also determined, with the finding that only tRNASerCAG (Ser1) has an Intron. At least five different types of tRNASerCAG genes exist in the genome of C.cylindracea. The nucleotide sequences of the flanking regions of these tRNASerCAG genes indicated that the tRNASerCAG gene has duplicated at least three times on the genome. The existence of multiple genes for tRNASerCAG on the genome may account for the observation that codon CUG is used very frequently in C.cylindracea. All of these tRNASerCAG genes contain the CCA sequence in their 3′ termini, suggesting the possibility that during their multiplication process In the evolution of the C.cylindracea genome, the tRNASerCAG molecule was Integrated into DNA via reverse transcription. © 1994 Oxford University Press.

    DOI PubMed CiNii

  • Unusual anticodon loop structure found in E.coli lysine tRNA

    Kimitsuna Watanabe, Nobuhiro Hayshi, Atsusi Oyama, Kazuya Nishikawa, Takuya Ueda, Kin-ichiro Miura

    Nucleic Acids Research   22 ( 1 ) 79 - 87  1994年01月

     概要を見る

    Although both tRNALya and tRNAGlu of tRNALya possess similar anticodon loop sequences, with the same hypermodifled nucleoside 5-methylaminomethyl-2-thiouridine (mnm582U) at the first position of their anticodons, the anticodon loop structures of these two tRNAs containing the modified nucleoside appear to be quite different as judged from the following observations. (1) The CD band derived from the mnm5s2U residue is negative for tRNAGlu, but positive for tRNALya. (2) The mnm5s2U monomer itself and the mnm5s2U-containing anticodon loop fragment of tRNALya show the same negative CD bands as that of tRNAGlu. (3) The positive CD band of tRNALya changes to negative when the temperature is raised. (4) The reactivity of the mnm5s2U residue toward H2O2 is much lower for tRNALya than for tRNAGlu. These features suggest that tRNALya has an unusual anticodon loop structure, in which the mnm5s2U residue takes a different conformation from that of tRNAGlu
    whereas the mnm5s2U base of tRNAGlu has no direct bonding with other bases and is accessible to a solvent, that of tRNALya exists as if in some way buried in its anticodon loop. The limited hydrolysis of both tRNAs by various RNases suggests that some differences exist in the higher order structures of tRNALya and tRNAGlu. The Influence of the unusual anticodon loop structure observed for tRNALya on its function in the translational process is also discussed. © 1994 Oxford University Press.

    DOI PubMed CiNii

  • UNIQUE STRUCTURE OF NEW SERINE TRANSFER-RNAS RESPONSIBLE FOR DECODING LEUCINE CODON CUG IN VARIOUS CANDIDA SPECIES AND THEIR PUTATIVE ANCESTRAL TRANSFER-RNA GENES

    T UEDA, T SUZUKI, T YOKOGAWA, K NISHIKAWA, K WATANABE

    BIOCHIMIE   76 ( 12 ) 1217 - 1222  1994年

     概要を見る

    In an asporogenic yeast, Candida cylindracea, codon CUG is not translated as leucine but as serine. On the basis of our recent work on the determination of the genetic code using in vitro translation systems coupled with isolation of the corresponding tRNA molecules, it appears that this non-universal genetic code is unitized not only in C cylindracea but also in various Hemiascomycetes. Here we show that in addition to the species already reported, three pathogenic yeasts, C guilliermondii, C lusitaniae and C tropicalis, have tRNA(Ser)CAG, indicating that this non-universal genetic code (CUG=Ser) also exists in these species. Determination of their primary structures revealed that the uridine conserved at position 33 in usual tRNAs, is replaced by guanosine or cytidine. This suggests that the three-dimensional structures of the anticodon loop of these tRNAs differ from the conventional structure comprising the U turn in this position. Moreover, we succeeded in isolating putative ancestral serine tRNA genes whose sequences are highly homologous to tRNA(Ser)CAG in each case. These tRNA genes all have the anticodon sequence CGA corresponding to the codon UCG, indicating that tRNA(Ser)CAG might have emerged from tRNA(Ser)CGA during evolutionary change of the assignment of codon CUG.

    DOI PubMed

  • CONFORMATIONAL PROPERTIES OF A NOVEL MODIFIED NUCLEOSIDE, 5-FORMYLCYTIDINE, FOUND AT THE FIRST POSITION OF THE ANTICODON OF BOVINE MITOCHONDRIAL TRNA(MET)

    G KAWAI, T YOKOGAWA, K NISHIKAWA, T UEDA, T HASHIZUME, JA MCCLOSKEY, S YOKOYAMA, K WATANABE

    NUCLEOSIDES & NUCLEOTIDES   13 ( 5 ) 1189 - 1199  1994年

     概要を見る

    Conformational properties of a novel modified nucleoside, 5-formylcytidine (f(5)C), which is found at the first position of the anticodon of bovine mitochondrial tRNA(Met), were analyzed by H-1-NMR spectroscopy. f(5)C has a normal amino tautomeric form at position 4 of the base moiety. The results indicate the presence of an intramolecular hydrogen bond between the carbonyl of the 5-formyl group and the 4-amino function. f(5)C was found to exhibit the C3'-endo conformation exclusively and the enthalpy difference (Delta H) between the C2'-endo and C3'-endo forms was found to be 1.56 +/- 0.13 kcal/mol, indicating f(5)C to be one of the most conformationally rigid nucleosides yet analyzed. The conformational rigidity of f(5)C may contribute to regulation of codon recognition by tRNA(Met).

    DOI CiNii

  • EXISTENCE OF NUCLEAR-ENCODED 5S-RIBOSOMAL-RNA IN BOVINE MITOCHONDRIA

    S YOSHIONARI, T KOIKE, T YOKOGAWA, K NISHIKAWA, T UEDA, K MIURA, K WATANABE

    FEBS LETTERS   338 ( 2 ) 137 - 142  1994年01月

     概要を見る

    A number of proteins functioning in mitochondria are synthesized in the cytoplasm and imported into the mitochondria via specific transport systems. In mammals, on the contrary, mitochondrial membranes have generally been considered to be impermeable to nucleic acids. However, here we show that an RNA with 120 nucleotides, the sequence of which is identical to that of the nuclear-encoded 5S RNA, exists in bovine mitochondria, although the mitochondrial genome encodes no 5S RNA gene. This RNA molecule was found to be retained in purified bovine mitochondria as well as in the mitoplasts, even after extensive treatment with an RNase, demonstrating that the 5S RNA is actually located inside the mitochondrial inner membrane. The 5S rRNA molecule was also shown to exist in mitochondria from rabbit and chicken.

    DOI PubMed CiNii

  • 遺伝暗号の起源と進化 変則暗号発生メカニズムについての一考察

    上田卓也, 横堀伸一, 渡辺公綱

    蛋白質 核酸 酵素   39 ( 15 )  1994年

    J-GLOBAL

  • 生物界における変則暗号とその発生機構

    上田 卓也, 渡辺 公綱

    蛋白質核酸酵素   38 ( 16 ) p2677 - 2691  1993年12月

    CiNii

  • NONUNIVERSAL DECODING OF THE LEUCINE CODON CUG IN SEVERAL CANDIDA SPECIES

    T OHAMA, T SUZUKI, M MORI, S OSAWA, T UEDA, K WATANABE, T NAKASE

    NUCLEIC ACIDS RESEARCH   21 ( 17 ) 4039 - 4045  1993年08月

     概要を見る

    It has been reported that CUG, a universal leucine codon, is read as serine in an asporogenic yeast, Candida cylindracea. The distribution of this non-universal genetic code in various yeast species was studied using an in vitro translation assay system with a synthetic messenger RNA containing CUG codons in-frame. It was found that CUG is used as a serine codon in six out of the fourteen species examined, while it is used for leucine in the remaining eight. The tRNA species responsible for the translation of codon CUG as serine was detected in all the six species in which CUG is translated as serine. The grouping according to the CUG codon assignments in these yeast species shows a good correlation with physiological classification by the chain lengths of the isoprenoid moiety of ubiquinone and the cell-wall sugar contained in the yeasts. The six Candida species examined in which CUG is used as serine belong to one distinct group in Hemiascomycetes.

    DOI

  • The evolution change of the genetic code as restricted by the anticodon and identity of transfer RNA

    Origin of Life and Evolution of the Biosphere   23,345  1993年

  • Codons AGA AGG are read as glycine in ascidian mitochondria

    J. Mol. Evol.   36 ( 1 ) 1 - 8  1993年

    DOI PubMed CiNii

  • 牛ミトコンドリアにおけるメチオニルtRNAのホルミル化反応とその翻訳における役割

    小池智浩, 竹本千重, 森谷二郎, 横川隆志, 上田卓也, SPREMULLI L L, 西川一八, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   16th  1993年

    J-GLOBAL

  • 原索類ミトコンドリアゲノムの遺伝子構造とAGRコドンの進化

    横堀伸一, 渡辺洋一, 横川隆志, 上島励, 上田卓也, 西川一八, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   16th  1993年

    J-GLOBAL

  • 軟体動物ミトコンドリアDNAの遺伝子配置とtRNAの異常構造

    海部雅之, 中西茂子, 山崎仁香, 貴家潤治, 横堀伸一, 上島励, 上田卓也, 西川一八, 渡辺公綱

    日本分子生物学会年会プログラム・講演要旨集   16th  1993年

    J-GLOBAL

  • SERINE TRANSFER-RNA COMPLEMENTARY TO THE NONUNIVERSAL SERINE CODON CUG IN CANDIDA-CYLINDRACEA - EVOLUTIONARY IMPLICATIONS

    T YOKOGAWA, T SUZUKI, T UEDA, M MORI, T OHAMA, Y KUCHINO, S YOSHINARI, MOTOKI, I, K NISHIKAWA, S OSAWA, K WATANABE

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   89 ( 16 ) 7408 - 7411  1992年08月

     概要を見る

    In the asporogenic yeast Candida cylindracea, the codon CUG is read as serine instead of leucine. This is an unusual instance in which the amino acid assignment of a codon deviates from the universal code. To infer the evolutionary process of this change, the tRNA with the anticodon sequence CAG, which is complementary to and thus responsible for translation of the codon CUG, has been identified. Indeed, this tRNA translates an in-frame CUG codon in a synthetic mRNA as serine in an in vitro translation system. The gene for the tRNA is interrupted by an intron in the anticodon loop. Sequence comparisons of the tRNA and its gene suggest that a single cytidine was inserted into the anticodon loop of the gene for tRNA(Ser)IGA during evolution to produce tRNA(Ser)CAG. The tRNA(Ser)CAG may be produced from its precursor molecule containing the cytidine insertion by splicing.

    DOI PubMed CiNii

  • THE T-LOOP REGION OF ANIMAL MITOCHONDRIAL TRANSFER RNA(SER)(AGY) IS A MAIN RECOGNITION SITE FOR HOMOLOGOUS SERYL-TRANSFER RNA-SYNTHETASE

    T UEDA, Y YOTSUMOTO, K IKEDA, K WATANABE

    NUCLEIC ACIDS RESEARCH   20 ( 9 ) 2217 - 2222  1992年05月

     概要を見る

    Recognition sites of bovine mitochondrial serine tRNA specific for codons AGY [tRNA(ser) (AGY)] by the cognate mitochondrial seryl-tRNA synthetase were studied using a range of tRNA(ser)(AGY) variants which were obtained by the in vitro transcription of synthetic tRNA genes with T7 RNA polymerase. Base replacements in the anticodon and discriminator sites did not affect serine acceptance. However, deletion and/or replacement in the T-loop region completely deprived the variants of their charging activities. Point mutation experiments in this region also showed that the adenosine residue in the middle of the T-loop (position 58), which is involved in tertiary interaction between the T-loop and the truncated D-arm [de Bruijn and Klug, 1983] played a significant role in the recognition process by the synthetase.

    DOI PubMed CiNii

  • RNA工学シリ-ズ-4-タンパク質合成におけるRNA修飾の役割

    上田 卓也, 渡辺 公綱

    バイオサイエンスとインダストリ-   50 ( 5 ) p433 - 437  1992年05月

    CiNii

  • A NOVEL CLOVERLEAF STRUCTURE FOUND IN MAMMALIAN MITOCHONDRIAL TRANSFER RNASER (UCN)

    T YOKOGAWA, Y WATANABE, Y KUMAZAWA, T UEDA, HIRAO, I, K MIURA, K WATANABE

    NUCLEIC ACIDS RESEARCH   19 ( 22 ) 6101 - 6105  1991年11月

     概要を見る

    Bovine mitochondrial tRNA(Ser)(UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated the bovine liver mitochondrial tRNA(Ser)(UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5'-terminal nucleotide and enzymatic determination of the whole sequence of tRNA(Ser)(UCN) revealed that the tRNA started from the third nucleotide of the putative tRNA(Ser)(UCN) gene, which had formerly been supposed. Enzymatic probing of tRNA(Ser)(UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNA(Ser)(UCN) genes of mammals such as human, rat and mouse, but not for those of non-mammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria.

    DOI PubMed CiNii

  • Phosphorothioate-containing RNAs show mRNA activity in the prokaryotic translation systems in vitro

    Takuya Ueda, Hideki Tohda, Nobutoshi Chikazumi, Fritz Eckstein, Kimitsuna Watanabe

    Nucleic Acids Research   19 ( 3 ) 547 - 552  1991年02月

     概要を見る

    Phosphorothioate-containing RNAs were generated by transcription of coliphage T7 ONA using the Sp diastereomers of ribonucleoside 5′-O-(1-thiotriphos-phates) and T7 RNA polymerase. RNAs In which a single nucleotide was substituted by the corresponding nucleoside phosphorothioate functioned as mRNA in the cell-free translation systems prepared from Escherichla coli and from an extreme theimophilic bacterium, Thermus thermophllus. This substitution increased the efficiency of protein synthesis by stabilizing the mRNAs in these systems. As the proportion of substituted nucleotides was increased, their mRNA activity was decreased accordingly. As judged from the analysis by SDS-polyacrylamide gel-electrophoresis, the proteins synthesized using phosphorothioate-containing mRNAs as template were identical to those obtained with unsubstituted mRNAs. However, larger proteins which were barely detectable when unsubstituted mRNA was used were well represented when phosphorothioate-RNA was used instead. The advantages in using the phosphorothioate-mRNAs in the in vitro translation systems are discussed. © 1991 Oxford University Press.

    DOI PubMed

  • ミトコンドリアRNAの研究--蛋白質合成系の研究

    上田 卓也

    旭硝子財団研究報告   ( 59 ) p235 - 241  1991年

    CiNii

  • CONVERSION OF AMINOACYLATION SPECIFICITY FROM TRANSFER RNA(TYR) TO TRANSFER RNA(SER) INVITRO

    H HIMENO, T HASEGAWA, T UEDA, K WATANABE, M SHIMIZU

    NUCLEIC ACIDS RESEARCH   18 ( 23 ) 6815 - 6819  1990年12月

     概要を見る

    The discrimination mechanism between tRNA(Ser) and tRNA(Tyr) wa studied using various in vitro transcripts of E. coli tRNA(Tyr) variants. The insertion of only two nucleotides into the variable stem of tRNA(Tyr) generates serine charging activity. The acceptor activities of some of the tRNA(Tyr) mutants with insertion sin the long variable arm were enhanced by changes in nucleotides at positions 9 and/or 20B, which are possible elements for dictating the orientation of the long variable arm. These findings suggest that the long variable arm is involved in recognition by seryl-tRNA synthetase in spite of sequence and length variations shown within tRNA(Ser) isoacceptors, and eventually serves as a determinant for selection from other tRNA species. Changing the anticodon from GUA to the serine antocodon GGA resulted in a marked decrease in tyrosine charging activity, but this mutant did not show any serine charging activity. The discriminator base, the fourth the base from the 3' end of tRNA, was also important for aminoacylation with tyrosine. Complete specificity change in vitro was facilitated by insertion of three nucleotides into the variable arm plus two nucleotide changes at positions 9 and 73.

    DOI PubMed

  • ROLE OF THE EXTRA G-C PAIR AT THE END OF THE ACCEPTOR STEM OF TRANSFER RNAHIS IN AMINOACYLATION

    H HIMENO, T HASEGAWA, T UEDA, K WATANABE, K MIURA, M SHIMIZU

    NUCLEIC ACIDS RESEARCH   17 ( 19 ) 7855 - 7863  1989年10月

    DOI PubMed CiNii

  • 第11回国際tRNAワ-クショップ見聞記

    上田 卓也, 渡辺 公綱

    蛋白質核酸酵素   30 ( 12 ) p1363 - 1368  1985年11月

    CiNii

  • LARGE-SCALE ISOLATION AND SOME PROPERTIES OF AGY-SPECIFIC SERINE TRANSFER-RNA FROM BOVINE HEART-MITOCHONDRIA

    T UEDA, T OHTA, K WATANABE

    JOURNAL OF BIOCHEMISTRY   98 ( 5 ) 1275 - 1284  1985年

  • STRUCTURAL FEATURES OF BOVINE MITOCHONDRIAL TRANSFER RNAAGY(SER) LACKING THE D-ARM

    T UEDA, K WATANABE, T OHTA

    PROCEEDINGS OF THE JAPAN ACADEMY SERIES B-PHYSICAL AND BIOLOGICAL SCIENCES   59 ( 10 ) 339 - 342  1983年

    DOI

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  • 基礎講義Ⅱ

    東京大学  

  • 分子生物学II

    東京大学  

 

学内研究所・附属機関兼任歴

  • 2022年
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    2024年

    理工学術院総合研究所   兼任研究員

特定課題制度(学内資金)

  • 人工細胞を指向したバイオシステムの無細胞合成系の開発

    2019年  

     概要を見る

    人工細胞の構築は合成生物学の目標の一つであり、再構築型無細胞タンパク質合成系PURE systemはその基幹システムとなることが期待されている。しかし、PURE systemを人工細胞へと発展させるためには、PURE systemが増殖し、またエネルギーを生産するシステムへと改良する必要がある。そのために、DNAからのリボソームの合成システム、転写系合成したtRNAによるPURE systemの構築、エネルギー生産細胞の合成、の三つのプロジェクトを推進し以下の成果を得た。大腸菌の小サブユニットについては、すでに個別精製したリボソームタンパク質とrRNAからの再構成に成功している。本課題では、リボソームタンパク質をDNAからPURE systemにより転写翻訳で合成し、rRNA存在下で30Sサブユニットとしてアセンブルさせ、その翻訳活性を解析した。転写合成するrRNAのanti-SD領域を人工配列に置換し、新規に再構成された30SとPURE system内の天然の30Sを区別した。解析の結果、新規に再構成した30Sサブユニットは翻訳活性を有することが示された。この結果は、Communications Biologyの投稿し受理された。また、50Sサブユニットについては、個別精製したリボソームタンパク質と23SrRNAと5Sからの再構成を行った。ショ糖密度勾配による超遠心による解析から再構成されていることが示され、また50Sサブユニットが活性は低いものの翻訳活性を有していることが示された。現在は、DNAから50Sサブユニットの合成を試みている。大腸菌の開始tRNAと20種類の伸長tRNAを試験管内転写系によって合成した。これらのtRNAは、天然のtRNAよりも活性は低いものの、翻訳活性を有していることが示された。この結果は、Nature Communicationに投稿し、現在formal peer review中である。転写rRNAに酵素的に修飾塩基を導入することで翻訳活性を向上させることに成功した。また個別の転写tRNAについてコドンの誤読度の評価を行い、高い忠実性を有していることが示された。バクテリオロドプシンとATP合成酵素をPUREで発現させたエネルギー(ATP)生産人工細胞の構築については、2019年3月にNature Communicationに発表している。このシステムはバクテリオロドプシンとATP合成酵素のF0部分のみをPURE systemでDNAから合成しリポソーム上に挿入したものであり、ATP合成酵素のF1部分はPURE systemでの発現量が不十分であり、天然のものを用いていた。本年度は、PURE systemの翻訳活性をさらに改善し、ATP合成酵素のF1部分もDNAからの合成が可能であることが示唆された。

  • 遺伝暗号の起源の解明の研究

    2019年  

     概要を見る

    遺伝暗号表の成立過程の解明は、生命の起源を解き明かすことである。本課題では、清水幹夫によって提唱されたtRNAのアンチコドンとディスクリミネーター塩基がC4Nコンプレックスを形成し、この複合体上のポケットでアミノ酸が認識・選択されることで遺伝暗号が生まれたとするC4N仮説を実証することを目的としている。いくつかのアミノ酸と対応するアンチコドンの分子模型を作製し、その相互作用を検討したところ、相互作用は可能であるが、水溶液中では、水分子の存在により容易に減弱される弱い相互作用であり、アミノ酸とRNAのみでは立体的な相互作用が困難であることが明らかとなった。この点については、水分子を排除し安定な環境を与える足場の分子を想定することで克服できると考えた。さまざまの分子を検討したが、以下の三点からリン酸のポリマー(ポリリン酸)が足場分子であるという作業仮説を持つに至った。①アミノ酸やヌクレオチドの脱水重合反応を促進しうる強い脱水分子である。②アンチコドンのリン酸部分に対して強く反発すること、また③アミノ酸のアミノ基を外側に固定すること、でアンチコドンの塩基部分とアミノ酸の相互作用を安定化しうる。さらに、現在のタンパク質合成系では、アミノ酸はATPのトリ(ポリ)リン酸部分の加水分解と共役してアミノ酸がAMPと共有結合を形成することで活性化されることを考えると、原始タンパク質合成系では、ポリリン酸は単なる安定化するのではなく触媒的に機能していた可能性がある。以上の考えに基づいて、ポリリン酸存在下での核酸によるアミノ酸の選択と重合からなる原始タンパク質合成系のモデルを構築した。ポリリン酸の生命の起源への関与を実験的に検討するために、現在さまざまな鎖長のポリリン酸を作製もしくは購入し、ポリリン酸による原始タンパク質合成系の実験的再現を進めている。これらのポリリン酸存在下でのアミノ酸の重合反応の最適条件を現在検討している。