A. Wali Karzai

SmpB, a unique RNA-binding protein essential for the peptide-tagging activity of SsrA (tmRNA).

In bacteria, SsrA RNA recognizes ribosomes stalled on defective messages and acts as a tRNA and mRNA to mediate the addition of a short peptide tag to the C‐terminus of the partially synthesized nascent polypeptide chain. The SsrA‐tagged protein is then degraded by C‐terminal‐specific proteases. SmpB, a unique RNA‐binding protein that is conserved throughout the bacterial kingdom, is shown here to be an essential component of the SsrA quality‐control system. Deletion of the smpB gene in Escherichia coli results in the same phenotypes observed in ssrA‐defective cells, including a variety of phage development defects and the failure to tag proteins translated from defective mRNAs. Purified SmpB binds specifically and with high affinity to SsrA RNA and is required for stable association of SsrA with ribosomes in vivo. Formation of an SmpB–SsrA complex appears to be critical in mediating SsrA activity after aminoacylation with alanine but prior to the transpeptidation reaction that couples this alanine to the nascent chain. SsrA RNA is present at wild‐type levels in the smpB mutant arguing against a model of SsrA action that involves direct competition for transcription factors.

RNase R degrades non‐stop mRNAs selectively in an SmpB‐tmRNA‐dependent manner

The SmpB‐tmRNA‐mediated trans‐translation system has two well‐established activities: rescuing ribosomes stalled on aberrant mRNAs and marking the associated protein fragments for proteolysis. Although the causative non‐stop mRNAs are known to be degraded, little is known about the enabling mechanism or the RNases involved in their disposal. We report that Escherichia coli has an enabling mechanism that requires RNase R activity and is dependent on the presence of SmpB protein and tmRNA, suggesting a requirement for active trans‐translation in facilitating RNase R engagement and promoting non‐stop mRNA decay. Interestingly, this selective transcript degradation by RNase R targets aberrant (non‐stop and multiple‐rare‐codon containing) mRNAs and does not affect the decay of related messages containing in‐frame stop codons. Most surprisingly, RNase II and PNPase do not play a significant role in tmRNA‐facilitated disposal of aberrant mRNAs. These findings demonstrate that RNase R is a crucial component of the trans‐translation‐mediated non‐stop mRNA decay process, thus providing a requisite activity well suited to complement the ribosome rescue and protein tagging functions of this unique quality control system.

Protein factors associated with the SsrA.SmpB tagging and ribosome rescue complex.

SsrA RNA acts as a tRNA and mRNA to modify proteins whose synthesis on ribosomes has stalled. Such proteins are marked for degradation by addition of peptide tags to their C termini in a reaction mediated by SsrA RNA and SmpB, a specific SsrA-RNA binding protein. Evidence is presented here for the existence of a larger ribonucleoprotein complex that contains ribosomal protein S1, phosphoribosyl pyrophosphate synthase, RNase R, and YfbG in addition to SsrA RNA and SmpB. Biochemical, genetic, and phylogenetic results suggest potential roles for some of these factors in various stages of the ribosome rescue and tagging process and/or the presence of functional interactions between one or more of these proteins and SsrA.

The highly conserved KEOPS/EKC complex is essential for a universal tRNA modification, t6A

The highly conserved Kinase, Endopeptidase and Other Proteins of small Size (KEOPS)/Endopeptidase‐like and Kinase associated to transcribed Chromatin (EKC) protein complex has been implicated in transcription, telomere maintenance and chromosome segregation, but its exact function remains unknown. The complex consists of five proteins, Kinase‐Associated Endopeptidase (Kae1), a highly conserved protein present in bacteria, archaea and eukaryotes, a kinase (Bud32) and three additional small polypeptides. We showed that the complex is required for a universal tRNA modification, threonyl carbamoyl adenosine (t6A), found in all tRNAs that pair with ANN codons in mRNA. We also showed that the bacterial ortholog of Kae1, YgjD, is required for t6A modification of Escherichia coli tRNAs. The ATPase activity of Kae1 and the kinase activity of Bud32 are required for the modification. The yeast protein Sua5 has been reported previously to be required for t6A synthesis. Using yeast extracts, we established an in vitro system for the synthesis of t6A that requires Sua5, Kae1, threonine, bicarbonate and ATP. It remains to be determined whether all reported defects of KEOPS/EKC mutants can be attributed to the lack of t6A, or whether the complex has multiple functions.

Lon Protease Degrades Transfer-Messenger RNA-Tagged Proteins

Bacterial trans translation is activated when translating ribosomes are unable to elongate or terminate properly. Small protein B (SmpB) and transfer-messenger RNA (tmRNA) are the two known factors required for and dedicated to trans translation. tmRNA, encoded by the ssrA gene, is a bifunctional molecule that acts both as a tRNA and as an mRNA during trans translation. The functions of tmRNA ensure that stalled ribosomes are rescued, the causative defective mRNAs are degraded, and the incomplete polypeptides are marked for targeted proteolysis. We present in vivo and in vitro evidence that demonstrates a direct role for the Lon ATP-dependent protease in the degradation of tmRNA-tagged proteins. In an endogenous protein tagging assay, lon mutants accumulated excessive levels of tmRNA-tagged proteins. In a reporter protein tagging assay with lambda-CI-N, the protein product of a nonstop mRNA construct designed to activate trans translation, lon mutant cells efficiently tagged the reporter protein, but the tagged protein exhibited increased stability. Similarly, a green fluorescent protein (GFP) construct containing a hard-coded C-terminal tmRNA tag (GFP-SsrA) exhibited increased stability in lon mutant cells. Most significantly, highly purified Lon preferentially degraded the tmRNA-tagged forms of proteins compared to the untagged forms. Based on these results, we conclude that Lon protease participates directly in the degradation of tmRNA-tagged proteins.

The smpB-ssrA Mutant of Yersinia pestis Functions as a Live Attenuated Vaccine To Protect Mice against Pulmonary Plague Infection

ABSTRACT The bacterial SmpB-SsrA system is a highly conserved translational quality control mechanism that helps maintain the translational machinery at full capacity. Here we present evidence to demonstrate that the smpB-ssrA genes are required for pathogenesis of Yersinia pestis, the causative agent of plague. We found that disruption of the smpB-ssrA genes leads to reduction in secretion of the type III secretion-related proteins YopB, YopD, and LcrV, which are essential for virulence. Consistent with these observations, the smpB-ssrA mutant of Y. pestis was severely attenuated in a mouse model of infection via both the intranasal and intravenous routes. Most significantly, intranasal vaccination of mice with the smpB-ssrA mutant strain of Y. pestis induced a strong antibody response. The vaccinated animals were well protected against subsequent lethal intranasal challenges with virulent Y. pestis. Taken together, our results indicate that the smpB-ssrA mutant of Y. pestis possesses the desired qualities for a live attenuated cell-based vaccine against pneumonic plague.

Non-stop mRNA decay initiates at the ribosome.

The translation machinery deciphers genetic information encoded within mRNAs to synthesize proteins needed for various cellular functions. Defective mRNAs that lack in‐frame stop codons trigger non‐productive stalling of ribosomes. We investigated how cells deal with such defective mRNAs, and present evidence to demonstrate that RNase R, a processive 3′‐to‐5′ exoribonuclease, is recruited to stalled ribosomes for the specific task of degrading defective mRNAs. The recruitment process is selective for non‐stop mRNAs and is dependent on the activities of SmpB protein and tmRNA. Most intriguingly, our analysis reveals that a unique structural feature of RNase R, the C‐terminal lysine‐rich (K‐rich) domain, is required both for productive ribosome engagement and targeted non‐stop mRNA decay activities of the enzyme. These findings provide new insights into how a general RNase is recruited to the translation machinery and highlight a novel role for the ribosome as a platform for initiating non‐stop mRNA decay.

Quality Control of Bacterial mRNA Decoding and Decay

Studies in eukaryotes and prokaryotes have revealed that gene expression is not only controlled through altering the rate of transcription but also through varying rates of translation and mRNA decay. Indeed, the expression level of a protein is strongly affected by the steady state level of its mRNA. RNA decay can, along with transcription, play an important role in regulating gene expression by fine-tuning the steady state level of a given transcript and affecting its subsequent decoding during translation. Alterations in mRNA stability can in turn have dramatic effects on cell physiology and as a consequence the fitness and survival of the organism. Recent evidence suggests that mRNA decay can be regulated in response to environmental cues in order to enable the organism to adapt to its changing surroundings. Bacteria have evolved unique post transcriptional control mechanisms to enact such adaptive responses through: 1) general mRNA decay, 2) differential mRNA degradation using small non-coding RNAs (sRNAs), and 3) selective mRNA degradation using the tmRNA quality control system. Here, we review our current understanding of these molecular mechanisms, gleaned primarily from studies of the model gram negative organism Escherichia coli, that regulate the stability and degradation of normal and defective transcripts.

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.

Francisella tularensis tmRNA system mutants are vulnerable to stress, avirulent in mice, and provide effective immune protection

Through targeted inactivation of the ssrA and smpB genes, we establish that the trans‐translation process is necessary for normal growth, adaptation to cellular stress and virulence by the bacterial pathogen Francisella tularensis. The mutant bacteria grow slower, have reduced resistance to heat and cold shocks, and are more sensitive to oxidative stress and sublethal concentrations of antibiotics. Modifications of the tmRNA tag and use of higher‐resolution mass spectrometry approaches enabled the identification of a large number of native tmRNA substrates. Of particular significance to understanding the mechanism of trans‐translation, we report the discovery of an extended tmRNA tag and extensive ladder‐like pattern of endogenous protein‐tagging events in F. tularensis that are likely to be a universal feature of tmRNA activity in eubacteria. Furthermore, the structural integrity and the proteolytic function of the tmRNA tag are both crucial for normal growth and virulence of F. tularensis. Significantly, trans‐translation mutants of F. tularensis are impaired in replication within macrophages and are avirulent in mouse models of tularemia. By exploiting these attenuated phenotypes, we find that the mutant strains provide effective immune protection in mice against lethal intradermal, intraperitoneal and intranasal challenges with the fully virulent parental strain.