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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Interferon β (IFN-β) is considered a signaling molecule with important therapeutic potential in cancer since IFN-β-induced gene transcription mediates antiproliferation and cell death induction. Whereas, TNF-related apoptosis inducing ligand/Apo2 ligand (TRAIL/Apo2L) has emerged as a promising anticancer agent because it induces apoptosis specifically in cancer cells. In this study, we elucidated that IFN-β augments TRAIL-induced apoptosis synergistically using five human malignant melanoma cells. All of these cells were induced apoptosis by TRAIL. Whereas, the response against IFN-β was different in amelanotic cells (A375 and CRL1579) and melanotic cells (G361, SK-MEL-28, and MeWo). The responsibility of amelanotic cells against IFN-β was higher than those of melanotic cells. The synergism of IFN-β and TRAIL were correlated with the responsibilities of the cells against IFN-β. The synergistic interaction was confirmed by a combination index based on the Chou-Talalay method. The upregulation of apoptosis in amelanotic cells was caused by very low doses of IFN-β (over 0.1 IU/ml). Both of p53-mediated intrinsic pathway and Fas-related extrinsic pathway were activated by IFN-β alone and combination with TRAIL. Further, TRAIL death receptors (DR4 and DR5) were upregulated by a low-dose IFN-β (over 0.1 IU/ml) and the expression was more promoted by the combination with TRAIL. It was clarified that the upregulation of DR5 is associated with the declination of viability.

Malignant melanoma is a highly aggressive skin cancer that is highly resistant to chemotherapy. Adjuvant therapy is administered to patients with melanoma that possess no microscopic metastases or have a high risk of developing microscopic metastases. Methylating agents, including dacarbazine (DTIC) and temozolomide (TMZ), pegylated interferon (IFN)-α2b and interleukin-2 have been approved for adjuvant immuno-chemotherapy; however, unsatisfactory results have been reported following the administration of methylating agents. IFN-β has been considered to be a signaling molecule with an important therapeutic potential in cancer. The aim of the present study was to elucidate whether antitumor effects could be augmented by the combination of TMZ and IFN-β in malignant melanoma. We evaluated the efficacy of TMZ and IFN-β by comparing O 6 -methylguanine-DNA transferase (MGMT)-proficient and -deficient cells, as MGMT has been reported to be associated with the resistance to methylating agents. Cell viability was determined by counting living cells with a Coulter counter, and apoptosis was analyzed by dual staining with Annexin V Alexa FluorR 488 and propidium iodide. The expression of proteins involved in the cell cycle, apoptosis and autophagy was evaluated by western blot analysis. The combined treatment with TMZ and IFN-β suppressed cell proliferation and induced cell cycle arrest. We also demonstrated that a combination of TMZ and IFN-β enhanced apoptosis and autophagy more efficiently compared with TMZ treatment alone. These findings suggest that antitumor activity may be potentiated by IFN-β in combination with TMZ.

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.

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.

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.

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.

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.

© 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We reported previously that our designed polypeptide 3 (21 residues), which has three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms not only an amphipathic -helix structure but also long fibrous assemblies in aqueous solution. To address the relationship between the electrical states of the polypeptide and its -helix and fibrous assembly formation, we characterized mutated polypeptides in which charged amino acid residues of 3 were replaced with Ser. We prepared the following polypeptides: 2S3 (LSTLAKA)(3), in which all Glu residues were replaced with Ser residues; 6S3 (LETLASA)(3), in which all Lys residues were replaced with Ser; and 2S6S3 (LSTLASA)(3); in which all Glu and Lys residues were replaced with Ser. In 0.1M KCl, 2S3 formed an -helix under basic conditions and 6S3 formed an -helix under acid conditions. In 1M KCl, they both formed -helices under a wide pH range. In addition, 2S3 and 6S3 formed fibrous assemblies under the same buffer conditions in which they formed -helices. -Helix and fibrous assembly formation by these polypeptides was reversible in a pH-dependent manner. In contrast, 2S6S3 formed an -helix under basic conditions in 1M KCl. Taken together, these findings reveal that the charge states of the charged amino acid residues and the charge state of the Leu residue located at the terminus play an important role in -helix formation.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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>

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

We present a new numerical path independent Ê-integral for calculating the stress intensity factors using a known auxiliary solution. The integral is path independent in the similar manner to the well known domain independent M-integral for calculating the stress intensity factors. The Ê-integral is, however, path independent even if the path contains any number of crack-tip and the integral may obtain the stress intensity factors at the onset of crack kinking by the path independent integral ; therefore, the integral path can be far from the crack-tip around which the numerical solution has noticeable error : The domain M-integral may not obtain the stress intensity factors at the onset of crack kinking and may be very difficult to obtain the stress intensity factors when there exist small cracks in the neighborhood of the crack-tip. For illustrative purposes, by using finite element method, numerical examples obtaining the stress intensity factors are presented for an extending straight crack and a kinking crack.

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.

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

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.

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.

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.

The ribosome display utilizes the formation of the mRNA-ribosome-polypeptide ternary complex in the cell-free protein synthesis system as the linking of the genotype (mRNA) to the phenotype (polypeptide). However, the presence of intrinsic components such as nucleases in the cell-extract based cell-free protein synthesis systems inevitably 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 Protein synthesis Using Recombinant Elements (PURE) system. The mRNA-ribosome-polypeptide ternary complex is highly stable in the PURE system and then the selected mRNA can be easily recovered, because activities of nucleases and other inhibitory factors are very low in the PURE system. Furthermore, omission of the release factors within the original PURE system can aid stalling of the ribosome at the termination codon to form the mRNA-ribosome-polypeptide ternary complex. We believe that these advantages assure the usability of the modified PURE system for ribosome display.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The CCA-adding enzyme [ATP (CTP): tRNA nucleotidyltransferase (EC. 2.7.7.25)] catalyzes the addition of the conserved CCA sequence to the 3'-terminus of tRNAs. It is known that CCA-adding enzymes from all three kingdoms recognize the elbow region of L-shape tRNA formed by the tertiary interaction between T-loop and D-loop. We here demonstrate that C. elegans mitochondrial CCA-adding enzyme has broad substrate specificity toward nematode mitochondrial tRNAs which lack entire T- or D-arms, by cloning and characterizing the recombinant protein.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 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.

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.

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.

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.

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.

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.

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).

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.

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).

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.