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.     

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.     

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.     

Small protein B interacts with the large and the small subunits of a stalled ribosome during trans-translation

During trans-translation, stalled bacterial ribosomes are rescued by small protein B (SmpB) and by transfer-messenger RNA (tmRNA). Stalled ribosomes switch translation from the defective messages to a short internal reading frame on tmRNA that tags the nascent peptide chain for degradation and recycles the ribosomes. We present evidences that SmpB binds the large and small ribosomal subunits in vivo and in vitro. The binding between SmpB and the ribosomal subunits is very tight, with a dissociation constant of 1.7 × 10⁻¹⁰ M, similar to its K_D for the 70S ribosome or for tmRNA. tmRNA displaces SmpB from its 50S binding but not from the 30S. In vivo, SmpB is detected on the 50S when trans-translation is impaired by lacking tmRNA or a functional SmpB. SmpB contacts the large subunit transiently and early during the trans-translational process. The affinity of SmpB for the two ribosomal subunits is modulated by tmRNA in the course of trans-translation. It is the first example of two copies of the same protein interacting with two different functional sites of the ribosomes.     

SsrA genes of streptomycetes and association of proteins to the tmRNA during development and cellular differentiation

Transfer-messenger RNA (tmRNA, 10Sa RNA, ssrA) is bacterial RNA having both tRNA and mRNA properties and playing an essential role in recycling of 70S ribosomes that are stalled on defective mRNA. The trans-translational system is thought to play a crucial role in bacterial survival under adverse conditions. Streptomycetes are Gram-positive soil bacteria exposed to various physical and chemical stresses that activate specialized responses such as synthesis of antibiotics and morphological differentiation. Comparative sequence analysis of ssrA genes of streptomycetes revealed the most significant differences in the central parts of tag-reading frames, in the stop codons and in the 15-34 nucleotide sequences following stop codons. A major challenge in understanding the interactions that control the function of tmRNA is the definition of protein interactions. Proteins from various phases of development of Streptomyces aureofaciens associated with tmRNA were analyzed. Using affinity chromatography on tmRNA-Sepharose and photo cross-linking experiments with [(32)P]labeled tmRNA seven proteins, the beta and beta'-subunits of DNA dependent RNA polymerase, polyribonucleotide nucleotidyltransferase (PNPase), ribosomal protein SS1, ATP-binding cassette transporters, elongation factor Tu, and SmpB were identified among the proteins associated with tmRNA of S. aureofaciens. We examined the functional role of ribosomal protein SS1 in a defined in vitro trans-translation system. Our data show that the protein SS1 that structurally differs from S1 of Escherichia coli is required for translation of the tmRNA tag-reading frame.     

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.     

<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.     

A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli

The bacterial ssrA gene codes for a dual function RNA, tmRNA, which possesses tRNA-like and mRNA-like regions. The tmRNA appends an oligopeptide tag to the polypeptide on the P-site tRNA by a trans-translation process that rescues ribosomes stalled on the mRNAs and targets the aberrant protein for degradation. In cells, processing of the stalled ribosomes is also pioneered by drop-off of peptidyl-tRNAs. The ester bond linking the peptide to tRNA is hydrolyzed by peptidyl-tRNA hydrolase (Pth), an essential enzyme, which releases the tRNA and the aberrant peptide. As the trans-translation mechanism utilizes the peptidyl-transferase activity of the stalled ribosomes to free the tRNA (as opposed to peptidyl-tRNA drop-off), the need for Pth to recycle such tRNAs is bypassed. Thus, we hypothesized that tmRNA may rescue a defect in Pth. Here, we show that overexpression of tmRNA rescues the temperature-sensitive phenotype of Escherichia coli (pth^ts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pth^ts strain. Consistent with our hypothesis, overexpression of tmRNA results in decreased accumulation of peptidyl-tRNA in E.coli. Furthermore, overproduction of tmRNA in E.coli strains deficient in ribosome recycling factor and/or lacking the release factor 3 enhances the rescue of pth^ts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.     

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.     

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 abundance in<Emphasis Type="Italic">Streptomyces aureofaciens,S. griseus</Emphasis> and<Emphasis Type="Italic">S. collinus</Emphasis> under stress-inducing conditions

tmRNA and protein SmpB are the main components required for rescue of stalled ribosomes incapable of properly elongating or terminating the polypeptide chain. We examined the tmRNA level and protein synthesis in Streptomyces aureofaciens, S. griseus and S. collinus synthesizing tetracycline, streptomycin and kirromycin, respectively, during various stress conditions. Downshift in temperature caused a decrease in protein synthesis but the level of tmRNA increased. Shift up in temperature induced decay of tmRNA in all strains and in S. collinus led to stimulation and in S. aureofaciens and S. griseus to inhibition of protein synthesis. At high NaCl concentrations protein synthesis was inhibited and tmRNA decayed. Shift in pH from 7.0 to 5.0 had no pronounced effect on the tmRNA level while upshift to pH 9.0 in S. collinus and S. aureofaciens caused inhibition of protein synthesis and decay of tmRNA in S. collinus. In contrast, protein synthesis and tmRNA level increased in S. griseus at the alkaline pH. Our data show that tmRNA abundance is important for survival of streptomycetes under certain unfavorable conditions.     

Emerging views on tmRNA-mediated protein tagging and ribosome rescue

Transfer-messenger RNA (tmRNA), also known as SsrA or 10Sa RNA, is a bacterial ribonucleic acid that recycles 70S ribosomes stalled on problematic messenger RNAs (mRNAs) and also contributes to the degradation of incompletely synthesized peptides. tmRNA acts initially as transfer RNA (tRNA), being aminoacylated at its 3'-end by alanyl-tRNA synthetase, to add alanine to the stalled polypeptide chain. Resumption of translation ensues not on the mRNA on which the ribosomes were stalled but at an internal position in tmRNA. Termination soon occurs, tmRNA recruiting the appropriate termination factors allowing the release of the tagged protein that is subsequently recognized and degraded by specific cytoplasmic and periplasmic proteases, and permits ribosome recycling. Recent data suggest that tmRNA tags bacterial proteins in three other instances; when ribosomes stall at internal sites; during 'readthrough' of canonical termination codons; and when ribosomes are at the termination codon of intact messages. The importance of bacterial tmRNAs for survival, growth under stress, and pathogenesis is also discussed. Recent in vivo and in vitro studies have identified novel ligands of tmRNA. Based on the available experimental evidences, an updated model of tmRNA mediated protein tagging and ribosome rescue in bacteria is presented.     

Escherichia coli tmRNA lacking pseudoknot 1 tags truncated proteins in vivo and in vitro

Transfer-messenger RNA (tmRNA) and protein SmpB facilitate trans-translation, a quality-control process that tags truncated proteins with short peptides recognized by a number of proteases and recycles ribosomes stalled at the 3′ end of mRNA templates lacking stop codons. The tmRNA molecule is a hybrid of tRNA- and mRNA-like domains that are usually connected by four pseudoknots (pk1–pk4). Replacement of pk1 with a single-stranded RNA yields pk1L, a mutant tmRNA that tags truncated proteins very poorly in vitro but very efficiently in vivo. However, deletion of the whole pk1 is deleterious for protein tagging. In contrast, deletion of helix 4 yields Δh4, a fully functional tmRNA derivative containing a single hairpin instead of pk1. Further deletions in the pk1 segment yield two subclasses of mutant tmRNAs that are unable to tag truncated proteins, but some of them bind to stalled ribosomes. Our studies demonstrate that pk1 is not essential for tmRNA functions but contributes to the stability of the tmRNA structure. Our studies also indicate that the length of this RNA segment is critical for both tmRNA binding to the ribosome and resumption of translation.     

Beyond ribosome rescue: tmRNA and co-translational processes

tmRNA is a unique bi-functional RNA that acts as both a tRNA and an mRNA to enter stalled ribosomes and direct the addition of a peptide tag to the C terminus of nascent polypeptides. Despite a reasonably clear understanding of tmRNA activity, the reason for its absolute conservation throughout the eubacteria is unknown. Although tmRNA plays many physiological roles in different bacterial systems, recent studies suggest a general role for trans-translation in monitoring protein folding and perhaps other co-translational processes. This review will focus on these new hypotheses and the data that support them.     

Structure and Function of tmRNA (10Sa RNA)

Modern data on the structure and function of transport/messenger (tm) RNA are reviewed. This stable RNA is involved in releasing ribosomes that are unable to complete protein synthesis on mRNA lacking the stop codon. The resulting abnormal proteins are rapidly degraded by specific proteases, which recognize a signal peptide encoded by the template region of tmRNA. The discovery of trans-translation has caused a particular interest in structural and functional studies of 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.     

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.