An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon–anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB''s affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.     

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.     

Functional SmpB-ribosome interactions require tmRNA

Small protein B (SmpB) is a requisite component of the transfer messenger RNA (tmRNA)-mediated bacterial translational quality control system known as trans-translation. The initial binding of tmRNA and its subsequent accommodation into the ribosomal A-site are activities intimately linked to SmpB protein function. From a mechanistic perspective, two key unanswered questions that require further investigation are: 1) what constitutes a stalled ribosome recognition complex and 2) does SmpB pre-bind ribosomes to recruit tmRNA. We have assessed, both in vivo and in vitro, the nature and stability of free SmpB interactions with stalled ribosomes and examined whether these interactions are functionally relevant. We present evidence to demonstrate that interaction of free SmpB with ribosomes is salt sensitive and significantly more labile than interaction of the SmpB.tmRNA complex with ribosomes. Upon dissociation of 70 S ribosomes SmpB partitions primarily with tmRNA rather than ribosomal subunits. This finding is consistent with biochemical and structural data demonstrating that tmRNA is the high-affinity binding partner of SmpB. Moreover, we show that under normal physiological conditions roughly similar numbers of SmpB and tmRNA molecules are present in cells. Our investigations also reveal that upon induction of a nonstop mRNA, SmpB is enriched in stalled ribosome fractions only in the presence of tmRNA. Based on these findings, we conclude that SmpB does not pre-bind stalled ribosome and that functional SmpB-stalled ribosome interactions require tmRNA. We propose that a 1:1:1 complex of SmpB.tmRNA.EF-Tu(GTP) recognizes and binds a stalled ribosome to initiate trans-translation.     

SmpB triggers GTP hydrolysis of elongation factor Tu on ribosomes by compensating for the lack of codon-anticodon interaction during trans-translation initiation

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.     

Ribosome hijacking: a role for small protein B during trans‐translation

Tight recognition of codon–anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the ‘tmRNA–SmpB’ system (transfer messenger RNA–small protein B). Remarkably, entry and accommodation of aminoacylated‐tmRNA into stalled ribosomes occur without a codon–anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon–anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for ΔsmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.     

Pre-binding of small protein B to a stalled ribosome triggers trans-translation

To rescue stalled ribosomes, eubacteria employ a molecule, transfer messenger RNA (tmRNA), which functions both as a tRNA and as an mRNA. With the help of small protein B (SmpB), tmRNA restarts protein synthesis and adds by the trans-translation mechanism a peptide tag to the stalled protein to target it for destruction by cellular proteases. Here, the cellular location and expression of endogenous SmpB were monitored in vivo. We report that SmpB is associated with 70S ribosomes and not in the soluble fraction, independently of the presence of tmRNA. In vitro, SmpB that is pre-bound to a stalled ribosome can trigger initiation of trans-translation. Our results demonstrate the existence of a novel pathway for the entry of tmRNA to the ribosome and for the trans-transfer of a nascent peptide chain from peptidyl-tRNA to charged tmRNA.     

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.     

Simultaneous and functional binding of SmpB and EF-Tu-TP to the alanyl acceptor arm of tmRNA.

Transfer-messenger RNA (tmRNA) mimics functions of aminoacyl-tRNA and mRNA, subsequently, when rescuing stalled ribosomes on a 3' truncated mRNA without stop codon in bacteria. In addition, this mechanism marks prematurely terminated proteins by a C-terminal peptide tag as a signal for degradation by specific cellular proteases. For Escherichia coli, previous studies on initial steps of this "trans-translation" mechanism revealed that tmRNA alanylation by Ala-tRNA synthetase and binding of Ala-tmRNA by EF-Tu-GTP for subsequent delivery to stalled ribosomes are inefficient when compared to analogous reactions with canonical tRNA(Ala). In other studies, protein SmpB and ribosomal protein S1 appeared to bind directly to tmRNA and to be indispensable for trans-translation. Here, we have searched for additional and synergistic effects of the latter two on tmRNA alanylation and its subsequent binding to EF-Tu-GTP. Kinetic analysis of functioning combined with band-shift experiments and structural probing demonstrate, that tmRNA may indeed form a multimeric complex with SmpB, S1 and EF-Tu-GTP, which leads to a considerably enhanced efficiency of the initial steps of trans-translation. Whereas S1 binds to the mRNA region of tmRNA, we have found that SmpB and EF-Tu both interact with its acceptor arm region. Interaction with SmpB and EF-Tu was also observed at the acceptor arm of Ala-tRNA(Ala), but there the alanylation efficiency was inhibited rather than stimulated by SmpB. Therefore, SmpB may function as an essential modulator of the tRNA-like acceptor arm of tmRNA during its successive steps in trans-translation.     

Independent binding sites of small protein B onto transfer-messenger RNA during trans-translation

Stalled bacterial ribosomes are freed by transfer-messenger RNA (tmRNA). With the help of small protein B (SmpB), protein synthesis restarts and tmRNA adds a tag to the stalled protein for destruction. The conformation of a 347 nt long tmRNA from a thermophile and its interactions with SmpB were monitored using structural probes. The RNA is highly folded, including the reading frame, with <30% of unpaired residues. Footprints between SmpB and tmRNA are in the elbow of the tRNA domain, in some pseudoknots including one essential for function and in the lower part of the stem exiting the tRNA domain. The footprints outside the tRNA domain are scattered onto the tmRNA sequence, but form a cluster onto its tertiary structure derived from cryo-EM data. Some footprints flank the first triplet to be translated in tmRNA, suggesting that SmpB participates in the insertion of the tmRNA-encoded reading frame into the decoding center. To discriminate between a conformational rearrangement of tmRNA and independent binding sites, surface plasmon resonance was used and has identified three independent binding sites of SmpB on the RNA, including the site on the tRNA domain. Accordingly, SmpB is proposed to move on the tmRNA scaffold during trans-translation.     

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.     

Active and Accurate trans-Translation Requires Distinct Determinants in the C-terminal Tail of SmpB Protein and the mRNA-like Domain of Transfer Messenger RNA (tmRNA)

Unproductive ribosome stalling in eubacteria is resolved by the actions of SmpB protein and transfer messenger (tm) RNA. We examined the functional significance of conserved regions of SmpB and tmRNA to the trans-translation process. Our investigations reveal that the N-terminal 20 residues of SmpB, which are located near the ribosomal decoding center, are dispensable for all known SmpB activities. In contrast, a set of conserved residues that reside at the junction between the tmRNA-binding core and the C-terminal tail of SmpB play an important role in tmRNA accommodation. Our data suggest that the highly conserved glycine 132 acts as a flexible hinge that enables movement of the C-terminal tail, thus permitting proper positioning and establishment of the tmRNA open reading frame (ORF) as the surrogate template. To gain further insights into the function of the SmpB C-terminal tail, we examined the tagging activity of hybrid variants of tmRNA and the SmpB protein, in which the tmRNA ORF or the SmpB C-terminal tail was substituted with the equivalent but highly divergent sequences from Francisella tularensis. We observed that the hybrid tmRNA was active but resulted in less accurate selection of the resume codon. Cognate hybrid SmpB was necessary to restore activity. Furthermore, accurate tagging was observed when the identity of the resume codon was reverted from GGC to GCA. Taken together, these data suggest that the engagement of the tmRNA ORF and the selection of the correct translation resumption point are distinct activities that are influenced by independent tmRNA and SmpB determinants.     

Single molecule imaging of the trans-translation entry process via anchoring of the tagged ribosome

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 × 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.     

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.     

A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation

In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by -1. We also found that U(85) is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U(85) from chemical modification.     

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.     

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。     

tmRNA and associated ligands: a puzzling relationship

Translation is an efficient and accurate mechanism, needing thorough systems of control-quality to ensure the correspondence between the information carried by the messenger RNA (mRNA) and the newly synthesized protein. Among them, trans-translation ensures delivering of stalled ribosomes when translation occurs on truncated mRNAs in bacteria, followed by the degradation of the incomplete nascent proteins. This process requires transfer-messenger RNA (tmRNA), an original molecule acting as both a tRNA and an mRNA. tmRNA first enters the decoding site of stuck ribosomes and, despite the lack of any codon-anticodon interaction, acts as a tRNA by transferring its alanine to the incomplete protein. Translation then switches to a small internal coding sequence (mRNA domain), which encodes a tag directing the incomplete protein towards degradation. Although playing a central role during trans-translation, tmRNA function depends on associated proteins. Genetic, biochemical and recent structural data are starting to unravel how the process takes place, by involving three main protein partners. Small protein B (SmpB) interacts with the tRNA-like domain (TLD) of tmRNA and is indispensable and specific to the process. Elongation factor Tu (EF-Tu) binds simultaneously the TLD and brings aminoacylated tmRNA to the ribosome, as for canonical tRNAs. Ribosomal protein S1 forms complexes with tmRNA, facilitating its recruitment by the stalled ribosomes. The chronology of events, however, is poorly understood and recent data shed light on the functions attributed to the proteins involved in trans-translation. This review focuses on the puzzling relationship that tmRNA has with these three protein ligands, putting forward trans-translation as a highly dynamical process.     

SmpB Contributes to Reading Frame Selection in the Translation of Transfer-Messenger RNA

Transfer-messenger RNA (tmRNA) acts first as a tRNA and then as an mRNA template to rescue stalled ribosomes in eubacteria. Together with its protein partner, SmpB (small protein B), tmRNA enters stalled ribosomes and transfers an Ala residue to the growing polypeptide chain. A remarkable step then occurs: the ribosome leaves the stalled mRNA and resumes translation using tmRNA as a template, adding a short peptide tag that destines the aborted protein for destruction. Exactly how the ribosome switches templates, resuming translation on tmRNA in the proper reading frame, remains unknown. Within the tmRNA sequence itself, five nucleotides (U85AGUC) immediately upstream of the first codon appear to direct frame selection. In particular, mutation of the conserved A86 results in severe loss of function both in vitro and in vivo. The A86C mutation causes translation to resume exclusively in the + 1 frame. Several candidate binding partners for this upstream sequence have been identified in vitro. Using a genetic selection for tmRNA activity in Escherichia coli, we identified mutations in the SmpB protein that restore the function of A86C tmRNA in vivo. The SmpB mutants increase tagging in the normal reading frame and reduce tagging in the + 1 frame. These results demonstrate that SmpB is functionally linked with the sequence upstream of the tmRNA template; both contribute to reading frame selection on tmRNA.     

tmRNA-SmpB: a journey to the centre of the bacterial ribosome

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans-translation quality control mechanism is activated by the transfer-messenger RNA bound to small protein B (tmRNA-SmpB ribonucleoprotein complex). Trans-translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo-electron microscopy structures of tmRNA-SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA-SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag-encoding sequence. This provides a structural basis for the transit of the large tmRNA-SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.     

Accommodation of tmRNA–SmpB into stalled ribosomes: A cryo-EM study

In eubacteria, translation of defective messenger RNAs (mRNAs) produces truncated polypeptides that stall on the ribosome. A quality control mechanism referred to as trans-translation is performed by transfer-messenger RNA (tmRNA), a specialized RNA acting as both a tRNA and an mRNA, associated with small protein B (SmpB). So far, a clear view of the structural movements of both the protein and RNA necessary to perform accommodation is still lacking. By using a construct containing the tRNA-like domain as well as the extended helix H2 of tmRNA, we present a cryo-electron microscopy study of the process of accommodation. The structure suggests how tmRNA and SmpB move into the ribosome decoding site after the release of EF-Tu·GDP. While two SmpB molecules are bound per ribosome in a preaccommodated state, our results show that during accommodation the SmpB protein interacting with the small subunit decoding site stays in place while the one interacting with the large subunit moves away. Relative to canonical translation, an additional movement is observed due to the rotation of H2. This suggests that the larger movement required to resume translation on a tmRNA internal open reading frame starts during accommodation.