掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press (OUP)  

Abstract

Proteins that exsert physiological functions during being translated have been discovered from prokaryotes to eukaryotes. These proteins, also called regulatory nascent chains, are common in interacting co-translationally with the ribosomes to stall them. In most cases, such a translational arrest is induced or released in response to changes in the intracellular environment. Cells take advantage of such an environmental sensitivity as a sensor to feedback-regulate gene expression. Recent studies reveal that certain nascent chains could also destabilize the translating ribosomes, leading to stochastic premature translation termination. In this review, we introduce several examples of bacterial nascent chain-based mechanisms of translation regulation by which bacteria regulate cellular functions.

DOI： 10.1093/jb/mvad007

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その他リンク： https://academic.oup.com/jb/article-pdf/173/4/227/50484123/mvad007.pdf

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Life depends on proteins, which all exist in nascent states when the growing polypeptide chain is covalently attached to a tRNA within the ribosome. Although the nascent chains, i.e. polypeptidyl-tRNAs (pep-tRNAs), are considered as merely transient intermediates during protein synthesis, recent advances have revealed that they are directly involved in a variety of cell functions, such as gene expression control. An increasing appreciation for fine-tuning at translational levels demands a general method to handle the pep-tRNAs on a large scale. Here, we developed a method termed peptidyl-tRNA enrichment using organic extraction and silica adsorption (PETEOS), and then identify their polypeptide moieties by mass spectrometry. As a proof-of-concept experiment using Escherichia coli, we identified ∼800 proteins derived from the pep-tRNAs, which were markedly biased towards the N-termini in the proteins, reflecting that PETEOS captured the intermediate pep-tRNA population during translation. Furthermore, we observed the changes in the pep-tRNA set in response to heat shock or antibiotic treatments. In summary, PETEOS will complement conventional methods to investigate nascent chains in the cell.

DOI： 10.1093/nar/gkac1276

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Robust translation elongation of any given amino acid sequence is required to shape proteomes. Nevertheless, nascent peptides occasionally destabilize ribosomes, since consecutive negatively charged residues in bacterial nascent chains can stochastically induce discontinuation of translation, in a phenomenon termed intrinsic ribosome destabilization (IRD). Here, using budding yeast and a human factor-based reconstituted translation system, we show that IRD also occurs in eukaryotic translation. Nascent chains enriched in aspartic acid (D) or glutamic acid (E) in their N-terminal regions alter canonical ribosome dynamics, stochastically aborting translation. Although eukaryotic ribosomes are more robust to ensure uninterrupted translation, we find many endogenous D/E-rich peptidyl-tRNAs in the N-terminal regions in cells lacking a peptidyl-tRNA hydrolase, indicating that the translation of the N-terminal D/E-rich sequences poses an inherent risk of failure. Indeed, a bioinformatics analysis reveals that the N-terminal regions of ORFs lack D/E enrichment, implying that the translation defect partly restricts the overall amino acid usage in proteomes.

DOI： 10.1038/s41467-022-35156-x

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担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Escherichia coli has multiple pathways to release nonproductive ribosome complexes stalled at the 3' end of nonstop mRNA: tmRNA (SsrA RNA)-mediated trans-translation and stop codon-independent termination by ArfA/RF2 or ArfB (YaeJ). The arfA mRNA lacks a stop codon and its expression is repressed by trans-translation. Therefore, ArfA is considered to complement the ribosome rescue activity of trans-translation, but the physiological situations in which ArfA is expressed have not been elucidated. Here, we found that the excision of CP4-57 prophage adjacent to E. coli ssrA leads to the inactivation of tmRNA and switches the primary rescue pathway from trans-translation to ArfA/RF2. This "rescue-switching" rearranges not only the proteome landscape in E. coli but also the phenotype such as motility. Furthermore, among the proteins with significantly increased abundance in the ArfA+ cells, we found ZntR, whose mRNA is transcribed together as the upstream part of nonstop arfA mRNA. Repression of ZntR and reconstituted model genes depends on the translation of the downstream nonstop ORFs that trigger the trans-translation-coupled exonucleolytic degradation by polynucleotide phosphorylase (PNPase). Namely, our studies provide a novel example of trans-translation-dependent regulation and re-define the physiological roles of prophage excision.

DOI： 10.1111/mmi.15003

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

The Escherichia coli chaperonin GroEL/ES (GroE) is one of the most extensively studied molecular chaperones. So far, ~80 proteins in E. coli are identified as GroE substrates that obligately require GroE for folding in vivo. In GroE-depleted cells, these substrates, when overexpressed, tend to form aggregates, whereas the GroE substrates expressed at low or endogenous levels are degraded, probably due to misfolded states. However, the protease(s) involved in the degradation process has not been identified. We conducted a mass-spectrometry-based proteomics approach to investigate the effects of three ATP-dependent proteases, Lon, ClpXP, and HslUV, on the E. coli proteomes under GroE-depleted conditions. A label-free quantitative proteomic method revealed that Lon protease is the dominant protease that degrades the obligate GroE substrates in the GroE-depleted cells. The deletion of DnaK/DnaJ, the other major E. coli chaperones, in the ∆lon strain did not cause major alterations in the expression or folding of the obligate GroE substrates, supporting the idea that the folding of these substrates is predominantly dependent on GroE.

DOI： 10.3390/molecules27123772

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担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Continuous translation elongation, irrespective of amino acid sequences, is a prerequisite for living organisms to produce their proteomes. However, nascent polypeptide products bear an inherent risk of elongation abortion. For example, negatively charged sequences with occasional intermittent prolines, termed intrinsic ribosome destabilization (IRD) sequences, weaken the translating ribosomal complex, causing certain nascent chain sequences to prematurely terminate translation. Here, we show that most potential IRD sequences in the middle of open reading frames remain cryptic and do not interrupt translation, due to two features of the nascent polypeptide. Firstly, the nascent polypeptide itself spans the exit tunnel, and secondly, its bulky amino acid residues occupy the tunnel entrance region, thereby serving as a bridge and protecting the large and small ribosomal subunits from dissociation. Thus, nascent polypeptide products have an inbuilt ability to ensure elongation continuity.

DOI： 10.15252/embj.2021108299

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Here we created artificial cells in which information of the genome of living cells is expressed by the elements encoded in the genome. We confirmed that the system works normally within artificial cells, which paves the way for reconstructing living cells from biomolecules.

DOI： 10.1039/d1cc04401j

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Aggregation is an inherent characteristic of proteins. Risk management strategies to reduce aggregation are critical for cells to survive upon stresses that induce aggregation. Cells cope with protein aggregation by utilizing a variety of chaperones, as exemplified by heat-shock proteins (Hsps). The heat stress-induced expression of IbpA and IbpB, small Hsps in Escherichia coli, is regulated by the σ32 heat-shock transcriptional regulator and the temperature-dependent translational regulation via mRNA heat fluctuation. We found that, even without heat stress, either the expression of aggregation-prone proteins or the ibpA gene deletion profoundly increases the expression of IbpA. Combined with other evidence, we propose novel mechanisms for the regulation of the small Hsps expression. Oligomeric IbpA self-represses the ibpA/ibpB translation, and mediates its own mRNA degradation, but the self-repression is relieved by sequestration of IbpA into the protein aggregates. Thus, the function of IbpA as a chaperone to form co-aggregates is harnessed as an aggregation sensor to tightly regulate the IbpA level. Since the excessive preemptive supply of IbpA in advance of stress is harmful, the prodigious and rapid expression of IbpA/IbpB on demand is necessary for IbpA to function as a first line of defense against acute protein aggregation.

DOI： 10.1111/mmi.14606

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Nascent polypeptides can modulate the polypeptide elongation speed on the ribosome. Here, we show that nascent chains can even destabilize the translating Escherichia coli ribosome from within. This phenomenon, termed intrinsic ribosome destabilization (IRD), occurs in response to a special amino acid sequence of the nascent chain, without involving the release or the recycling factors. Typically, a consecutive array of acidic residues and those intermitted by alternating prolines induce IRD. The ribosomal protein bL31, which bridges the two subunits, counteracts IRD, such that only strong destabilizing sequences abort translation in living cells. We found that MgtL, the leader peptide of a Mg2+ transporter (MgtA), contains a translation-aborting sequence, which sensitizes the ribosome to a decline in Mg2+ concentration and thereby triggers the MgtA-upregulating genetic scheme. Translation proceeds at an inherent risk of ribosomal destabilization, and nascent chain-ribosome complexes can function as a Mg2+ sensor by harnessing IRD.

DOI： 10.1016/j.molcel.2017.10.020

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study, Escherichia coli SecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members of E. coli that were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl-tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

DOI： 10.1073/pnas.1520560113

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記述言語：日本語  

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3' end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3' end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding.

DOI： 10.1093/nar/gku1069

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Translation terminates at stop codon. Without stop codon, ribosome cannot terminate translation properly and reaches and stalls at the 3'-end of the mRNA lacking stop codon. Bacterial tmRNA-mediated trans-translation releases such stalled ribosome and targets the protein product to degradation by adding specific "degradation tag." Recently two alternative ribosome rescue factors, ArfA (YhdL) and ArfB (YaeJ), have been found in Escherichia coli. These three ribosome rescue systems are different each other in terms of molecular mechanism of ribosome rescue and their activity, but they are mutually related and co-operate to maintain the translation system in shape. This suggests the biological significance of ribosome rescue.

DOI： 10.3389/fmicb.2014.00156

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

The ribosomes stalled at the end of non-stop mRNAs must be rescued for productive cycles of cellular protein synthesis. Escherichia coli possesses at least three independent mechanisms that resolve non-productive translation complexes (NTCs). While tmRNA (SsrA) mediates trans-translation to terminate translation, ArfA (YhdL) and ArfB (YaeJ) induce hydrolysis of ribosome-tethered peptidyl-tRNAs. ArfB is a paralogue of the release factors (RFs) and directly catalyses the peptidyl-tRNA hydrolysis within NTCs. In contrast, the mechanism of the ArfA action had remained obscure beyond its ability to bind to the ribosome. Here, we characterized the ArfA pathway of NTC resolution in vitro and identified RF2 as a factor that cooperates with ArfA to hydrolyse peptidyl-tRNAs located in the P-site of the stalled ribosome. This reaction required the GGQ (Gly-Gly-Gln) hydrolysis motif, but not the SPF (Ser-Pro-Phe) codon-recognition sequence, of RF2 and was stimulated by tRNAs. From these results we suggest that ArfA binds to the vacant A-site of the stalled ribosome with possible aid from association with a tRNA, and then recruits RF2, which hydrolyses peptidyl-tRNA in a GGQ motif-dependent but codon-independent manner. In support of this model, the ArfA-RF2 pathway did not act on the SecM-arrested ribosome, which contains an aminoacyl-tRNA in the A-site.

DOI： 10.1111/j.1365-2958.2012.08190.x

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Accumulation of stalled ribosomes at the 3' end of mRNA without a stop codon (non-stop mRNA) is supposed to be toxic to bacterial cells. Escherichia coli has at least two distinct systems to rescue such stalled ribosomes: SsrA-dependent trans-translation and ArfA-dependent ribosome rescue. Combination of the ssrA and arfA mutations is synthetically lethal, suggesting the significance of ribosome rescue. In this study, we identified the E. coli yaeJ gene, encoding a peptide-release factor homologue with GGQ motif, as a multicopy suppressor of the lethal phenotype of ssrA arfA double mutant. The YaeJ protein was shown to bind to ribosomes. Both in vivo and in vitro, YaeJ showed the ribosome-rescue activity and promoted the hydrolysis of peptidyl-tRNA residing in the stalled ribosome. Missense mutation in the GGQ motif or deletion of the C-terminal unstructured tail abolished both the suppressor activity for ssrA arfA synthetic lethality and the ribosome-rescue activity, suggesting the importance of these structural features. On the basis of these observations, we propose that YaeJ acts as a stop codon-independent peptidyl-tRNA hydrolysing factor through binding to ribosomes stalled at the 3' end of non-stop mRNAs. It was also suggested that ArfA and YaeJ rescue the stalled ribosomes by distinct mechanisms.

DOI： 10.1111/j.1365-2958.2011.07607.x

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ribosomes translating mRNA without an in-frame stop codon (non-stop mRNA) stall at its 3' end. In eubacteria, such ribosomes are rescued by SsrA-mediated trans-translation. Recently, we have shown that Escherichia coli ArfA (formerly YhdL) also rescues stalled ribosomes by a mechanism distinct from that of trans-translation. Synthetic lethality phenotype of ssrA arfA double mutants suggests that accumulation of stalled ribosomes is deleterious to E. coli cells. In this report, we show that the expression of ArfA is tightly regulated by the system involving trans-translation. Both premature transcription termination and specific cleavage by RNase III were programmed at the specific sites within the arfA open reading frame (ORF) and produced arfA non-stop mRNA. C-terminally truncated ArfA protein synthesized from arfA non-stop mRNA was tagged through SsrA-mediated trans-translation and degraded in wild type cell. In the absence of SsrA, however, C-terminally truncated ArfA escaped from degradation and had a function to rescue stalled ribosomes. Full-length ArfA produced only when arfA mRNA escapes from both premature transcription termination and RNase III cleavage was unstable. From these results, we illustrate a regulatory model in which ArfA is expressed only when it is needed, namely, when the ribosome rescue activity of trans-translation system is insufficient to support cell viability. This sophisticated regulatory mechanism suggests that the ArfA-mediated ribosome rescue is a backup system for trans-translation.

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although co-translational biological processes attract much attention, no general and easy method has been available to detect cellular nascent polypeptide chains, which we propose to call collectively a "nascentome." We developed a method to selectively detect polypeptide portions of cellular polypeptidyl-tRNAs and used it to study the generality of the quality control reactions that rescue dead-end translation complexes. To detect nascent polypeptides, having their growing ends covalently attached to a tRNA, cellular extracts are separated by SDS-PAGE in two dimensions, first with the peptidyl-tRNA ester bonds preserved and subsequently after their in-gel cleavage. Pulse-labeled nascent polypeptides of Escherichia coli form a characteristic line below the main diagonal line, because each of them had contained a tRNA of nearly uniform size in the first-dimension electrophoresis but not in the second-dimension. The detection of nascent polypeptides, separately from any translation-completed polypeptides or degradation products thereof, allows us to follow their fates to gain deeper insights into protein biogenesis and quality control pathways. It was revealed that polypeptidyl-tRNAs were significantly stabilized in E. coli upon dysfunction of the tmRNA-ArfA ribosome-rescuing system, whose function had only been studied previously using model constructs. Our results suggest that E. coli cells are intrinsically producing aberrant translation products, which are normally eliminated by the ribosome-rescuing mechanisms.

DOI： 10.1371/journal.pone.0028413

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

Although SsrA(tmRNA)-mediated trans-translation is thought to maintain the translation capacity of bacterial cells by rescuing ribosomes stalled on messenger RNA lacking an in-frame stop codon, single disruption of ssrA does not crucially hamper growth of Escherichia coli. Here, we identified YhdL (renamed ArfA for alternative ribosome-rescue factor) as a factor essential for the viability of E. coli in the absence of SsrA. The ssrA-arfA synthetic lethality was alleviated by SsrA(DD) , an SsrA variant that adds a proteolysis-refractory tag through trans-translation, indicating that ArfA-deficient cells require continued translation, rather than subsequent proteolysis of the truncated polypeptide. In accordance with this notion, depletion of SsrA in the ΔarfA background led to reduced translation of a model protein without affecting transcription, and puromycin, a codon-independent mimic of aminoacyl-tRNA, rescued the bacterial growth under such conditions. That ArfA takes over the role of SsrA was suggested by the observation that its overexpression enabled detection of the polypeptide encoded by a model non-stop mRNA, which was otherwise SsrA-tagged and degraded. In vitro, purified ArfA acted on a ribosome-nascent chain complex to resolve the peptidyl-tRNA. These results indicate that ArfA rescues the ribosome stalled at the 3' end of a non-stop mRNA without involving trans-translation.

DOI： 10.1111/j.1365-2958.2010.07375.x

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記述言語：英語   掲載種別：研究発表ペーパー・要旨（国際会議）   出版者・発行元：GENETICS SOC JAPAN  

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開催年月日： 2024年3月13日 - 2024年3月14日

記述言語：日本語   会議種別：口頭発表（一般）  

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