Resolving Nonstop Translation Complexes Is a Matter of Life or Death

Problems during gene expression can result in a ribosome that has translated to the 3′ end of an mRNA without terminating at a stop codon, forming a nonstop translation complex. The nonstop translation complex contains a ribosome with the mRNA and peptidyl-tRNA engaged, but because there is no codon in the A site, the ribosome cannot elongate or terminate the nascent chain. Recent work has illuminated the importance of resolving these nonstop complexes in bacteria. Transfer-messenger RNA (tmRNA)-SmpB specifically recognizes and resolves nonstop translation complexes in a reaction known as trans-translation. trans-Translation releases the ribosome and promotes degradation of the incomplete nascent polypeptide and problematic mRNA. tmRNA and SmpB have been found in all bacteria and are essential in some species. However, other bacteria can live without trans-translation because they have one of the alternative release factors, ArfA or ArfB. ArfA recruits RF2 to nonstop translation complexes to promote hydrolysis of the peptidyl-tRNAs. ArfB recognizes nonstop translation complexes in a manner similar to tmRNA-SmpB recognition and directly hydrolyzes the peptidyl-tRNAs to release the stalled ribosomes. Genetic studies indicate that most or all species require at least one mechanism to resolve nonstop translation complexes. Consistent with such a requirement, small molecules that inhibit resolution of nonstop translation complexes have broad-spectrum antibacterial activity. These results suggest that resolving nonstop translation complexes is a matter of life or death for bacteria.     

Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA

Messenger RNAs lacking a stop codon trap ribosomes at their 3′ ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3′ extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3′ extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3′ extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.     

Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons

During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNA(Arg4) upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.     

<Emphasis Type="Italic">Trans</Emphasis>-translation: Findings and hypotheses

Trans-translation is a unique process that switches the synthesis of a polypeptide hain encoded by a nonstop mRNA to the mRNA-like domain of tmRNA. The process is used in bacterial cells for rescuing the ribosomes arrested during translation of nonstop mRNA and directing this mRNA and the polypeptide product for degradation. Activity of tmRNA is essential for bacterial survival under adverse conditions, the quality control of translation, and the regulation of certain physiological pathways. The review focuses on recent advances in trans-translation studies. Details of the tmRNA-SmpB interaction and the structures of early ribosomal complexes are characterized, the causes of the appearance of an empty A site in the translating ribosome and possible mechanisms of the stalled ribosome recognition and resume codon determination are discussed, and the proteins degrading nonstop mRNAs and tagged peptides are considered.     

Prophage excision switches the primary ribosome rescue pathway and rescue-associated gene regulations in Escherichia coli

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.     

YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl-tRNA on stalled ribosomes

In bacteria, ribosomes often become stalled and are released by a trans-translation process mediated by transfer-messenger RNA (tmRNA). In the absence of tmRNA, however, there is evidence that stalled ribosomes are released from non-stop mRNAs. Here, we show a novel ribosome rescue system mediated by a small basic protein, YaeJ, from Escherichia coli, which is similar in sequence and structure to the catalytic domain 3 of polypeptide chain release factor (RF). In vitro translation experiments using the E. coli-based reconstituted cell-free protein synthesis system revealed that YaeJ can hydrolyze peptidyl–tRNA on ribosomes stalled by both non-stop mRNAs and mRNAs containing rare codon clusters that extend downstream from the P-site and prevent Ala-tmRNA•SmpB from entering the empty A-site. In addition, YaeJ had no effect on translation of a normal mRNA with a stop codon. These results suggested a novel tmRNA-independent rescue system for stalled ribosomes in E. coli. YaeJ was almost exclusively found in the 70S ribosome and polysome fractions after sucrose density gradient sedimentation, but was virtually undetectable in soluble fractions. The C-terminal basic residue-rich extension was also found to be required for ribosome binding. These findings suggest that YaeJ functions as a ribosome-attached rescue device for stalled ribosomes.     

tmRNA Is Essential in Shigella flexneri

Nonstop mRNAs pose a challenge for bacteria, because translation cannot terminate efficiently without a stop codon. The trans-translation pathway resolves nonstop translation complexes by removing the nonstop mRNA, the incomplete protein, and the stalled ribosome. P1 co-transduction experiments demonstrated that tmRNA, a key component of the trans-translation pathway, is essential for viability in Shigella flexneri. tmRNA was previously shown to be dispensable in the closely related species Escherichia coli, because E. coli contains a backup system for trans-translation mediated by the alternative release factor ArfA. Genome sequence analysis showed that S. flexneri does not have a gene encoding ArfA. E. coli ArfA could suppress the requirement for tmRNA in S. flexneri, indicating that tmRNA is essential in S. flexneri because there is no functional backup system. These data suggest that resolution of nonstop translation complexes is required for most bacteria.     

tmRNA·SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes

Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 Å resolution structure of the tmRNA·SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA·SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.     

ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue

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.     

ArfA Recruits RF2 into Stalled Ribosomes

During translation in Escherichia coli, the ribosome rescue factor YaeJ and the alternative ribosome rescue factor (ArfA, previously called YhdL) can release stalled ribosomes from mRNA. Here, I used a reconstituted cell-free protein synthesis system to examine YaeJ- and ArfA-dependent recycling of stalled ribosomes, in which mRNA lacks in-frame stop codons. It is shown that YaeJ alone could recycle the ribosome but that ArfA required the presence of release factor 2 (RF2). Furthermore, I show that RF2 binds to stalled ribosomes only in the presence of ArfA, demonstrating that ArfA recruits RF2 into the A site of the ribosome to facilitate peptidyl-tRNA hydrolysis. It is also demonstrated that the efficiency of the ArfA-dependent process decreases rapidly with an increase in mRNA length downstream of the A site of the ribosome whereas YaeJ function is maintained on mRNA with sufficient length. From the results, I discuss differences of in vivo roles of these two systems in addition to the well-known tmRNA-dependent trans-translation system.     

Interaction of SmpB with ribosome from directed hydroxyl radical probing

To add a tag-peptide for degradation to the nascent polypeptide in a stalled ribosome, an unusual translation called trans-translation is facilitated by transfer-messenger RNA (tmRNA) having an upper half of the tRNA structure and the sequence encoding the tag-peptide except the first alanine. During this event, tmRNA enters the vacant A-site of the stalled ribosome without a codon–anticodon interaction, but with a protein factor SmpB. Here, we studied the sites and modes of binding of SmpB to the ribosome by directed hydroxyl radical probing from Fe(II) tethered to SmpB variants. It revealed two SmpB-binding sites, A-site and P-site, on the ribosome. Each SmpB can be superimposed on the lower half of tRNA behaving in translation. The sites of cleavages from Fe(II) tethered to the C-terminal residues of A-site SmpB are aligned along the mRNA path towards the downstream tunnel, while those of P-site SmpB are found almost exclusively around the region of the codon–anticodon interaction in the P-site. We propose a new model of trans-translation in that the C-terminal tail of SmpB initially recognizes the decoding region and the mRNA path free of mRNA by mimicking mRNA.     

Distinct tmRNA sequence elements facilitate RNase R engagement on rescued ribosomes for selective nonstop mRNA decay

trans-Translation, orchestrated by SmpB and tmRNA, is the principal eubacterial pathway for resolving stalled translation complexes. RNase R, the leading nonstop mRNA surveillance factor, is recruited to stalled ribosomes in a trans-translation dependent process. To elucidate the contributions of SmpB and tmRNA to RNase R recruitment, we evaluated Escherichia coli–Francisella tularensis chimeric variants of tmRNA and SmpB. This evaluation showed that while the hybrid tmRNA supported nascent polypeptide tagging and ribosome rescue, it suffered defects in facilitating RNase R recruitment to stalled ribosomes. To gain further insights, we used established tmRNA and SmpB variants that impact distinct stages of the trans-translation process. Analysis of select tmRNA variants revealed that the sequence composition and positioning of the ultimate and penultimate codons of the tmRNA ORF play a crucial role in recruiting RNase R to rescued ribosomes. Evaluation of defined SmpB C-terminal tail variants highlighted the importance of establishing the tmRNA reading frame, and provided valuable clues into the timing of RNase R recruitment to rescued ribosomes. Taken together, these studies demonstrate that productive RNase R-ribosomes engagement requires active trans-translation, and suggest that RNase R captures the emerging nonstop mRNA at an early stage after establishment of the tmRNA ORF as the surrogate mRNA template.     

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem–loop in tmRNA during later steps of trans-translation.     

反式翻译模型

反式翻译是细菌体内一种修复翻译水平上受阻的遗传信息表达过程的机制。tmRNA是反式翻译的核心分子,它兼具tRNA和mRNA的特点,在SmpB蛋白的帮助下特异性识别携带mRNA缺失体的核糖体,在核糖体蛋白S1的传递作用下结合在A位点上,一方面延续被中断的mRNA上的遗传信息,一方面终止蛋白质的合成,释放被束缚的核糖体和tRNA进入新的翻译过程。本文对近年来关于反式翻译模型的研究进行综述。     

In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation

Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation.     

SmpB functions in various steps of trans-translation

tmRNA has a dual function as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA’s tag sequence. SmpB is a tmRNA binding protein that has been identified to be essential for trans-translation in vivo. To further study the function of SmpB, an S30 fraction from an Escherichia coli strain, in which the set of genes for SmpB and tmRNA has been deleted from the genome, and His-tagged SmpB active in trans-translation were prepared. The SmpB-depleted S30 fraction had an ability to facilitate poly(U)-dependent tag-peptide synthesis in vitro when purified His-tagged SmpB was exogenously added together with tmRNA, although SmpB was not required for in vitro poly(U)-dependent poly(Phe) synthesis. It was also found that depletion of SmpB leads to a decrease in the level of tmRNA in the cell. In addition, SmpB considerably enhanced the aminoacylation of tmRNA by alanyl-tRNA synthetase in vitro. The aminoacylation enhancement by SmpB, the binding of SmpB to tmRNA and the effect of depletion of SmpB on the expression level of tmRNA in the cell were all affected by some mutations in the tRNA-like domain which cause a defect in ribosome binding leading to a trans-translation deficiency. These results demonstrate that, via binding to the tRNA-like domain of tmRNA, SmpB plays various roles: rescuing the tmRNA molecule from degradation in the cell, enhancing the aminoacylation of tmRNA and mediating the binding of tmRNA to ribosome.     

ArfA recruits release factor 2 to rescue stalled ribosomes by peptidyl‐tRNA hydrolysis in Escherichia coli

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.     

Structural features of the tmRNA–ribosome interaction

Trans-translation is a process which switches the synthesis of a polypeptide chain encoded by a nonstop messenger RNA to the mRNA-like domain of a transfer-messenger RNA (tmRNA). It is used in bacterial cells for rescuing the ribosomes arrested during translation of damaged mRNA and directing this mRNA and the product polypeptide for degradation. The molecular basis of this process is not well understood. Earlier, we developed an approach that allowed isolation of tmRNA–ribosomal complexes arrested at a desired step of tmRNA passage through the ribosome. We have here exploited it to examine the tmRNA structure using chemical probing and cryo-electron microscopy tomography. Computer modeling has been used to develop a model for spatial organization of the tmRNA inside the ribosome at different stages of trans-translation.     

Bifunctional transfer-messenger RNA

Transfer-messenger RNA (tmRNA) is a bifunctional RNA that has properties of a tRNA and an mRNA. tmRNA uses these two functions to release ribosomes stalled during translation and target the nascent polypeptides for degradation. This concerted reaction, known as trans-translation, contributes to translational quality control and regulation of gene expression in bacteria. tmRNA is conserved throughout bacteria, and is one of the most abundant RNAs in the cell, suggesting that trans-translation is of fundamental importance for bacterial fitness. Mutants lacking tmRNA activity typically have severe phenotypes, including defects in viability, virulence, and responses to environmental stresses.