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1.  Ribosome Rescue and Translation Termination at Non-Standard Stop Codons by ICT1 in Mammalian Mitochondria 
PLoS Genetics  2014;10(9):e1004616.
Release factors (RFs) govern the termination phase of protein synthesis. Human mitochondria harbor four different members of the class 1 RF family: RF1Lmt/mtRF1a, RF1mt, C12orf65 and ICT1. The homolog of the essential ICT1 factor is widely distributed in bacteria and organelles and has the peculiar feature in human mitochondria to be part of the ribosome as a ribosomal protein of the large subunit. The factor has been suggested to rescue stalled ribosomes in a codon-independent manner. The mechanism of action of this factor was obscure and is addressed here. Using a homologous mitochondria system of purified components, we demonstrate that the integrated ICT1 has no rescue activity. Rather, purified ICT1 binds stoichiometrically to mitochondrial ribosomes in addition to the integrated copy and functions as a general rescue factor, i.e. it releases the polypeptide from the peptidyl tRNA from ribosomes stalled at the end or in the middle of an mRNA or even from non-programmed ribosomes. The data suggest that the unusual termination at a sense codon (AGA/G) of the oxidative-phosphorylation enzymes CO1 and ND6 is also performed by ICT1 challenging a previous model, according to which RF1Lmt/mtRF1a is responsible for the translation termination at non-standard stop codons. We also demonstrate by mutational analyses that the unique insertion sequence present in the N-terminal domain of ICT1 is essential for peptide release rather than for ribosome binding. The function of RF1mt, another member of the class1 RFs in mammalian mitochondria, was also examined and is discussed.
Author Summary
Mammalian mitochondrial ICT1, a bacterial ArfB homolog, is interestingly an integral component of the mitoribosome (MRPL58). The mechanism of ribosome rescue by this factor was obscure and is addressed here. Utilizing a homologous mitochondria system of purified components we demonstrate that the integrated ICT1 has no rescue activity, as opposed to a previous model. Rather, purified ICT1 added to mitoribosomes has a general rescue activity; it recycles ribosomes stalled at the end or in the middle of mRNAs and can even hydrolyze peptidyl-tRNA bound to non-programmed ribosomes. These results further imply that ICT1 can function in the translation termination at non-standard stop codons AGA/G in mammalian mitochondria. Our data challenge a previous model claiming that RF1Lmt/mtRF1a is responsible for the translation termination at non-standard stop codons. A mutational study indicates that the unique insertion sequence in ICT1 is essential for peptide release. The function of RF1mt, another member of the class1 RFs in mammalian mitochondria, was also examined and is discussed.
doi:10.1371/journal.pgen.1004616
PMCID: PMC4169044  PMID: 25233460
2.  Evolution and Diversification of the Organellar Release Factor Family 
Molecular Biology and Evolution  2012;29(11):3497-3512.
Translation termination is accomplished by proteins of the Class I release factor family (RF) that recognize stop codons and catalyze the ribosomal release of the newly synthesized peptide. Bacteria have two canonical RFs: RF1 recognizes UAA and UAG, RF2 recognizes UAA and UGA. Despite that these two release factor proteins are sufficient for de facto translation termination, the eukaryotic organellar RF protein family, which has evolved from bacterial release factors, has expanded considerably, comprising multiple subfamilies, most of which have not been functionally characterized or formally classified. Here, we integrate multiple sources of information to analyze the remarkable differentiation of the RF family among organelles. We document the origin, phylogenetic distribution and sequence structure features of the mitochondrial and plastidial release factors: mtRF1a, mtRF1, mtRF2a, mtRF2b, mtRF2c, ICT1, C12orf65, pRF1, and pRF2, and review published relevant experimental data. The canonical release factors (mtRF1a, mtRF2a, pRF1, and pRF2) and ICT1 are derived from bacterial ancestors, whereas the others have resulted from gene duplications of another release factor. These new RF family members have all lost one or more specific motifs relevant for bona fide release factor function but are mostly targeted to the same organelle as their ancestor. We also characterize the subset of canonical release factor proteins that bear nonclassical PxT/SPF tripeptide motifs and provide a molecular-model-based rationale for their retained ability to recognize stop codons. Finally, we analyze the coevolution of canonical RFs with the organellar genetic code. Although the RF presence in an organelle and its stop codon usage tend to coevolve, we find three taxa that encode an RF2 without using UGA stop codons, and one reverse scenario, where mamiellales green algae use UGA stop codons in their mitochondria without having a mitochondrial type RF2. For the latter, we put forward a “stop-codon reinvention” hypothesis that involves the retargeting of the plastid release factor to the mitochondrion.
doi:10.1093/molbev/mss157
PMCID: PMC3472500  PMID: 22688947
release factor; translation termination; mitochondrion; plastid; evolution; genetic code
3.  mtRF1a Is a Human Mitochondrial Translation Release Factor Decoding the Major Termination Codons UAA and UAG 
Molecular Cell  2007;27(5):745-757.
Summary
Human mitochondria contain their own genome, encoding 13 polypeptides that are synthesized within the organelle. The molecular processes that govern and facilitate this mitochondrial translation remain unclear. Many key factors have yet to be characterized—for example, those required for translation termination. All other systems have two classes of release factors that either promote codon-specific hydrolysis of peptidyl-tRNA (class I) or lack specificity but stimulate the dissociation of class I factors from the ribosome (class II). One human mitochondrial protein has been previously identified in silico as a putative member of the class I release factors. Although we could not confirm the function of this factor, we report the identification of a different mitochondrial protein, mtRF1a, that is capable in vitro and in vivo of terminating translation at UAA/UAG codons. Further, mtRF1a depletion in HeLa cells led to compromised growth in galactose and increased production of reactive oxygen species.
doi:10.1016/j.molcel.2007.06.031
PMCID: PMC1976341  PMID: 17803939
RNA
4.  A functional peptidyl-tRNA hydrolase, ICT1, has been recruited into the human mitochondrial ribosome 
The EMBO Journal  2010;29(6):1116-1125.
Bioinformatic analysis classifies the human protein encoded by immature colon carcinoma transcript-1 (ICT1) as one of a family of four putative mitochondrial translation release factors. However, this has not been supported by any experimental evidence. As only a single member of this family, mtRF1a, is required to terminate the synthesis of all 13 mitochondrially encoded polypeptides, the true physiological function of ICT1 was unclear. Here, we report that ICT1 is an essential mitochondrial protein, but unlike the other family members that are matrix-soluble, ICT1 has become an integral component of the human mitoribosome. Release-factor assays show that although ICT1 has retained its ribosome-dependent PTH activity, this is codon-independent; consistent with its loss of both domains that promote codon recognition in class-I release factors. Mutation of the GGQ domain common to ribosome-dependent PTHs causes a loss of activity in vitro and, crucially, a loss of cell viability, in vivo. We suggest that ICT1 may be essential for hydrolysis of prematurely terminated peptidyl-tRNA moieties in stalled mitoribosomes.
doi:10.1038/emboj.2010.14
PMCID: PMC2845271  PMID: 20186120
mitoribosomes; peptidyl-tRNA hydrolase; translation release factor
5.  Ribosome Recycling Factor and Release Factor 3 Action Promotes TnaC-Peptidyl-tRNA Dropoff and Relieves Ribosome Stalling during Tryptophan Induction of tna Operon Expression in Escherichia coli▿  
Journal of Bacteriology  2007;189(8):3147-3155.
Upon tryptophan induction of tna operon expression in Escherichia coli, the leader peptidyl-tRNA, TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document}, resists cleavage, resulting in ribosome stalling at the tnaC stop codon. This stalled ribosome blocks Rho factor binding and action, preventing transcription termination in the tna operon's leader region. Plasmid-mediated overexpression of tnaC was previously shown to inhibit cell growth by reducing uncharged \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document} availability. Which factors relieve ribosome stalling, facilitate TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document} cleavage, and relieve growth inhibition were addressed in the current study. In strains containing the chromosomal tna operon and lacking a tnaC plasmid, the overproduction of ribosome recycling factor (RRF) and release factor 3 (RF3) reduced tna operon expression. Their overproduction in vivo also increased the rate of cleavage of TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document}, relieving the growth inhibition associated with plasmid-mediated tnaC overexpression. The overproduction of elongation factor G or initiation factor 3 did not have comparable effects, and tmRNA was incapable of attacking TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document} in stalled ribosome complexes. The stability of TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document} was increased appreciably in strains deficient in RRF and RF3 or deficient in peptidyl-tRNA hydrolase. These findings reveal the existence of a natural mechanism whereby an amino acid, tryptophan, binds to ribosomes that have just completed the synthesis of TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document}. Bound tryptophan inhibits RF2-mediated cleavage of TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document}, resulting in the stalling of the ribosome translating tnaC mRNA. This stalling results in increased transcription of the structural genes of the tna operon. RRF and RF3 then bind to this stalled ribosome complex and slowly release TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document}. This release allows ribosome recycling and permits the cleavage of TnaC-\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}tRNA_{2}^{Pro}\end{equation*}\end{document} by peptidyl-tRNA hydrolase.
doi:10.1128/JB.01868-06
PMCID: PMC1855834  PMID: 17293419
6.  YaeJ is a novel ribosome-associated protein in Escherichia coli that can hydrolyze peptidyl–tRNA on stalled ribosomes 
Nucleic Acids Research  2010;39(5):1739-1748.
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.
doi:10.1093/nar/gkq1097
PMCID: PMC3061065  PMID: 21051357
7.  A physiological connection between tmRNA and peptidyl-tRNA hydrolase functions in Escherichia coli 
Nucleic Acids Research  2004;32(20):6028-6037.
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 (pthts). Conversely, a null mutation in ssrA enhances the temperature-sensitive phenotype of the pthts 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 pthts strains. We discuss the physiological relevance of these observations to highlight a major role of tmRNA in decreasing cellular peptidyl-tRNA load.
doi:10.1093/nar/gkh924
PMCID: PMC534616  PMID: 15547251
8.  Kinetics of paused ribosome recycling in Escherichia coli 
Journal of molecular biology  2009;394(2):251-267.
Summary
The bacterial tmRNA•SmpB system recycles stalled translation complexes in a process termed ‘ribosome rescue’. tmRNA•SmpB specifically recognizes ribosomes that are paused at or near the 3′ end of truncated mRNA, and therefore nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA•SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA+ cells (T1/2 = 22 ± 2.2 s), compared to ΔtmRNA cells (T1/2 = 32 ± 1.6 s). Overexpression of release factor-1 (RF-1) greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA+ and ΔtmRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA•SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t1/2 = 61 ± 7.3 s) in ΔtmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor (RRF) in ΔtmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA•SmpB activity is rate-limited by mRNA cleavage, and that RF-3 and RRF do not constitute a tmRNA-independent rescue pathway as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in ΔtmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway.
doi:10.1016/j.jmb.2009.09.020
PMCID: PMC2783371  PMID: 19761774
peptidyl-tRNA; release factor; ribosome pausing; tmRNA; translation termination
9.  Isolation of Translating Ribosomes Containing Peptidyl-tRNAs for Functional and Structural Analyses 
Recently, structural and biochemical studies have detailed many of the molecular events that occur in the ribosome during inhibition of protein synthesis by antibiotics and during nascent polypeptide synthesis. Some of these antibiotics, and regulatory nascent polypeptides mostly in the form of peptidyl-tRNAs, inhibit either peptide bond formation or translation termination1-7. These inhibitory events can stop the movement of the ribosome, a phenomenon termed "translational arrest". Translation arrest induced by either an antibiotic or a nascent polypeptide has been shown to regulate the expression of genes involved in diverse cellular functions such as cell growth, antibiotic resistance, protein translocation and cell metabolism8-13. Knowledge of how antibiotics and regulatory nascent polypeptides alter ribosome function is essential if we are to understand the complete role of the ribosome in translation, in every organism.
Here, we describe a simple methodology that can be used to purify, exclusively, for analysis, those ribosomes translating a specific mRNA and containing a specific peptidyl-tRNA14. This procedure is based on selective isolation of translating ribosomes bound to a biotin-labeled mRNA. These translational complexes are separated from other ribosomes in the same mixture, using streptavidin paramagnetic beads (SMB) and a magnetic field (MF). Biotin-labeled mRNAs are synthesized by run-off transcription assays using as templates PCR-generated DNA fragments that contain T7 transcriptional promoters. T7 RNA polymerase incorporates biotin-16-UMP from biotin-UTP; under our conditions approximately ten biotin-16-UMP molecules are incorporated in a 600 nt mRNA with a 25% UMP content. These biotin-labeled mRNAs are then isolated, and used in in vitro translation assays performed with release factor 2 (RF2)-depleted cell-free extracts obtained from Escherichia coli strains containing wild type or mutant ribosomes. Ribosomes translating the biotin-labeled mRNA sequences are stalled at the stop codon region, due to the absence of the RF2 protein, which normally accomplishes translation termination. Stalled ribosomes containing the newly synthesized peptidyl-tRNA are isolated and removed from the translation reactions using SMB and an MF. These beads only bind biotin-containing messages.
The isolated, translational complexes, can be used to analyze the structural and functional features of wild type or mutant ribosomal components, or peptidyl-tRNA sequences, as well as determining ribosome interaction with antibiotics or other molecular factors 1,14-16. To examine the function of these isolated ribosome complexes, peptidyl-transferase assays can be performed in the presence of the antibiotic puromycin1. To study structural changes in translational complexes, well established procedures can be used, such as i) crosslinking to specific amino acids14 and/or ii) alkylation protection assays1,14,17.
doi:10.3791/2498
PMCID: PMC3197406  PMID: 21403627
10.  Visualizing the transfer-messenger RNA as the ribosome resumes translation 
The EMBO Journal  2010;29(22):3819-3825.
Visualizing the transfer-messenger RNA as the ribosome resumes translation
Bacterial ribosomes that are stalled on mRNAs lacking a stop codon can be rescued by a process called ‘transtranslation' that involves the ribonucleoprotein complex tmRNA–SmpB. This cryo-EM study, and the copublished study by Weis et al, reveal how translation on tmRNA is resumed
Bacterial ribosomes stalled by truncated mRNAs are rescued by transfer-messenger RNA (tmRNA), a dual-function molecule that contains a tRNA-like domain (TLD) and an internal open reading frame (ORF). Occupying the empty A site with its TLD, the tmRNA enters the ribosome with the help of elongation factor Tu and a protein factor called small protein B (SmpB), and switches the translation to its own ORF. In this study, using cryo-electron microscopy, we obtained the first structure of an in vivo-formed complex containing ribosome and the tmRNA at the point where the TLD is accommodated into the ribosomal P site. We show that tmRNA maintains a stable ‘arc and fork' structure on the ribosome when its TLD moves to the ribosomal P site and translation resumes on its ORF. Based on the density map, we built an atomic model, which suggests that SmpB interacts with the five nucleotides immediately upstream of the resume codon, thereby determining the correct selection of the reading frame on the ORF of tmRNA.
doi:10.1038/emboj.2010.255
PMCID: PMC2989109  PMID: 20940705
tmRNA; trans-translation; SmpB
11.  ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue 
Nucleic Acids Research  2014;42(21):13339-13352.
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
PMCID: PMC4245945  PMID: 25355516
12.  Overcoming stalled translation in human mitochondria 
Protein synthesis is central to life and maintaining a highly accurate and efficient mechanism is essential. What happens when a translating ribosome stalls on a messenger RNA? Many highly intricate processes have been documented in the cytosol of numerous species, but how does organellar protein synthesis resolve this stalling issue? Mammalian mitochondria synthesize just thirteen highly hydrophobic polypeptides. These proteins are all integral components of the machinery that couples oxidative phosphorylation. Consequently, it is essential that stalled mitochondrial ribosomes can be efficiently recycled. To date, there is no evidence to support any particular molecular mechanism to resolve this problem. However, here we discuss the observation that there are four predicted members of the mitochondrial translation release factor family and that only one member, mtRF1a, is necessary to terminate the translation of all thirteen open reading frames in the mitochondrion. Could the other members be involved in the process of recycling stalled mitochondrial ribosomes?
doi:10.3389/fmicb.2014.00374
PMCID: PMC4103422  PMID: 25101074
mitochondria; release factor; ICT1; ribosome rescue; ribosome stalling; protein synthesis; translation
13.  Competition between trans-translation and termination or elongation of translation 
Nucleic Acids Research  2005;33(17):5544-5552.
The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3′-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3′-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.
doi:10.1093/nar/gki871
PMCID: PMC1243801  PMID: 16204455
14.  Resolving Nonstop Translation Complexes Is a Matter of Life or Death 
Journal of Bacteriology  2014;196(12):2123-2130.
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.
doi:10.1128/JB.01490-14
PMCID: PMC4054194  PMID: 24706739
15.  Regulation of mtrF Expression in Neisseria gonorrhoeae and Its Role in High-Level Antimicrobial Resistance 
Journal of Bacteriology  2005;187(11):3713-3720.
The obligate human pathogen Neisseria gonorrhoeae uses the MtrC-MtrD-MtrE efflux pump to resist structurally diverse hydrophobic antimicrobial agents (HAs), some of which bathe mucosal surfaces that become infected during transmission of gonococci. Constitutive high-level HA resistance occurs by the loss of a repressor (MtrR) that negatively controls transcription of the mtrCDE operon. This high-level HA resistance also requires the product of the mtrF gene, which is located downstream and transcriptionally divergent from mtrCDE. MtrF is a putative inner membrane protein, but its role in HA resistance mediated by the MtrC-MtrD-MtrE efflux pump remains to be determined. High-level HA resistance can also be mediated through an induction process that requires enhanced transcription of mtrCDE when gonococci are grown in the presence of a sublethal concentration of Triton X-100. We now report that inactivation of mtrF results in a significant reduction in the induction of HA resistance and that the expression of mtrF is enhanced when gonococci are grown under inducing conditions. However, no effect was observed on the induction of mtrCDE expression in an MtrF-negative strain. The expression of mtrF was repressed by MtrR, the major repressor of mtrCDE expression. In addition to MtrR, another repressor (MpeR) can downregulate the expression of mtrF. Repression of mtrF by MtrR and MpeR was additive, demonstrating that the repressive effects mediated by these regulators are independent processes.
doi:10.1128/JB.187.11.3713-3720.2005
PMCID: PMC1112036  PMID: 15901695
16.  tmRNA-mediated trans-translation as the major ribosome rescue system in a bacterial cell 
Transfer messenger RNA (tmRNA; also known as 10Sa RNA or SsrA RNA) is a small RNA molecule that is conserved among bacteria. It has structural and functional similarities to tRNA: it has an upper half of the tRNA-like structure, its 5’ end is processed by RNase P, it has typical tRNA-specific base modifications, it is aminoacylated with alanine, it binds to EF-Tu after aminoacylation and it enters the ribosome with EF-Tu and GTP. However, tmRNA lacks an anticodon, and instead it has a coding sequence for a short peptide called tag-peptide. An elaborate interplay of actions of tmRNA as both tRNA and mRNA with the help of a tmRNA-binding protein, SmpB, facilitates trans-translation, which produces a single polypeptide from two mRNA molecules. Initially alanyl-tmRNA in complex with EF-Tu and SmpB enters the vacant A-site of the stalled ribosome like aminoacyl-tRNA but without a codon–anticodon interaction, and subsequently truncated mRNA is replaced with the tag-encoding region of tmRNA. During these processes, not only tmRNA but also SmpB structurally and functionally mimics both tRNA and mRNA. Thus trans-translation rescues the stalled ribosome, thereby allowing recycling of the ribosome. Since the tag-peptide serves as a target of AAA+ proteases, the trans-translation products are preferentially degraded so that they do not accumulate in the cell. Although alternative rescue systems have recently been revealed, trans-translation is the only system that universally exists in bacteria. Furthermore, it is unique in that it employs a small RNA and that it prevents accumulation of non-functional proteins from truncated mRNA in the cell. It might play the major role in rescuing the stalled translation in the bacterial cell.
doi:10.3389/fgene.2014.00066
PMCID: PMC3985003  PMID: 24778639
tmRNA; SmpB; ribosome; trans-translation; molecular mimicry
17.  Structural Basis for the Rescue of Stalled Ribosomes: Structure of YaeJ Bound to the Ribosome 
Science (New York, N.Y.)  2012;335(6074):1370-1372.
In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom–resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNAifMet and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.
doi:10.1126/science.1217443
PMCID: PMC3377438  PMID: 22422986
18.  Ribosome regulation by the nascent peptide. 
Microbiological Reviews  1996;60(2):366-385.
Studies of bacterial and eukaryotic systems have identified two-gene operons in which the translation product of the upstream gene influences translation of the downstream gene. The upstream gene, referred to as a leader (gene) in bacterial systems or an upstream open reading frame (uORF) in eukaryotes, encodes a peptide that interferes with a function(s) of its translating ribosome. The peptides are therefore cis-acting negative regulators of translation. The inhibitory peptides typically consist of fewer than 25 residues and function prior to emergence from the ribosome. A biological role for this class of translation inhibitor is demonstrated in translation attenuation, a form or regulation that controls the inducible translation of the chloramphenicol resistance genes cat and cmlA in bacteria. Induction of cat or cmlA requires ribosome stalling at a particular codon in the leader region of the mRNA. Stalling destabilizes an adjacent, downstream mRNA secondary structure that normally sequesters the ribosome-binding site for the cat or cmlA coding regions. Genetic studies indicate that the nascent, leader-encoded peptide is the selector of the site of ribosome stalling in leader mRNA by cis interference with translation. Synthetic leader peptides inhibit ribosomal peptidyltransferase in vitro, leading to the prediction that this activity is the basis for stall site selection. Recent studies have shown that the leader peptides are rRNA-binding peptides with targets at the peptidyl transferase center of 23S rRNA. uORFs associated with several eukaryotic genes inhibit downstream translation. When inhibition depends on the specific codon sequence of the uORF, it has been proposed that the uORF-encoded nascent peptide prevents ribosome release from the mRNA at the uORF stop codon. This sets up a blockade to ribosome scanning which minimizes downstream translation. Segments within large proteins also appear to regulate ribosome activity in cis, although in most of the known examples the active amino acid sequences function after their emergence from the ribosome, cis control of translation by the nascent peptide is gene specific; nearly all such regulatory peptides exert no obvious trans effects in cells. The in vitro biochemical activities of the cat/cmla leader peptides on ribosomes and rRNA suggest a mechanism through which the nascent peptide can modify ribosome behavior. Other cis-acting regulatory peptides may involve more complex ribosomal interactions.
PMCID: PMC239448  PMID: 8801438
19.  Sequestration of specific tRNA species cognate to the last sense codon of an overproduced gratuitous protein 
Nucleic Acids Research  2000;28(23):4725-4732.
High-level expression of non-functional model proteins, derived from elongation factor EF-Tu by the deletion of an essential domain, greatly inhibits the growth of Escherichia coli partly deficient in peptidyl-tRNA hydrolase. High-level expression in wild-type cells has little effect on growth. The inhibitory effect is therefore presumably due to the sequestration of essential tRNA species, partly in the form of free peptidyl-tRNA. The growth inhibitory effect can be modulated by changing the last sense codon in the genes encoding the model proteins. Thus, replacement of Ser by Lys or His at this position increases growth inhibition. The effects of 11 changes studied are related to the rates of accumulation previously observed of the corresponding families of peptidyl-tRNA. Two non-exclusive hypotheses are proposed to account for these observations: first, the last sense codon of mRNA is a prefered site of peptidyl-tRNA drop-off in cells, due to the slow rate of translation termination compared with sense codon translation; secondly, the relatively long pause of the ribosome at the stop codon (of the order of 1 s), results in significant temporary sequestration on the ribosome of the tRNA cognate to the last sense codon.
PMCID: PMC115180  PMID: 11095683
20.  The role of upstream sequences in selecting the reading frame on tmRNA 
BMC Biology  2008;6:29.
Background
tmRNA acts first as a tRNA and then as an mRNA to rescue stalled ribosomes in eubacteria. Two unanswered questions about tmRNA function remain: how does tmRNA, lacking an anticodon, bypass the decoding machinery and enter the ribosome? Secondly, how does the ribosome choose the proper codon to resume translation on tmRNA? According to the -1 triplet hypothesis, the answer to both questions lies in the unique properties of the three nucleotides upstream of the first tmRNA codon. These nucleotides assume an A-form conformation that mimics the codon-anticodon interaction, leading to recognition by the decoding center and choice of the reading frame. The -1 triplet hypothesis is important because it is the most credible model in which direct binding and recognition by the ribosome sets the reading frame on tmRNA.
Results
Conformational analysis predicts that 18 triplets cannot form the correct structure to function as the -1 triplet of tmRNA. We tested the tmRNA activity of all possible -1 triplet mutants using a genetic assay in Escherichia coli. While many mutants displayed reduced activity, our findings do not match the predictions of this model. Additional mutagenesis identified sequences further upstream that are required for tmRNA function. An immunoblot assay for translation of the tmRNA tag revealed that certain mutations in U85, A86, and the -1 triplet sequence result in improper selection of the first codon and translation in the wrong frame (-1 or +1) in vivo.
Conclusion
Our findings disprove the -1 triplet hypothesis. The -1 triplet is not required for accommodation of tmRNA into the ribosome, although it plays a minor role in frame selection. Our results strongly disfavor direct ribosomal recognition of the upstream sequence, instead supporting a model in which the binding of a separate ligand to A86 is primarily responsible for frame selection.
doi:10.1186/1741-7007-6-29
PMCID: PMC2481249  PMID: 18590561
21.  SecM-Stalled Ribosomes Adopt an Altered Geometry at the Peptidyl Transferase Center 
PLoS Biology  2011;9(1):e1000581.
A structure of a ribosome stalled during translation of the SecM peptide provides insight into the mechanism by which the large subunit active site is inactivated.
As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA–nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNAPro in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.
Author Summary
In all cells, ribosomes perform the job of making proteins. As the proteins are synthesized they pass through a tunnel in the ribosome, and some growing proteins interact with the tunnel, leading to stalling of protein synthesis. Here, we used cryo-electron microscopy to determine the structure of a ribosome stalled during the translation of the Escherichia coli secretion monitor (SecM) polypeptide chain. The structure reveals the path of the SecM peptide through the tunnel as well as the sites of interaction with the tunnel components. Interestingly, the structure shows a shift in the position of the transfer RNA (tRNA) to which the growing SecM polypeptide chain is attached. Since peptide bond formation during protein synthesis requires precise placement of the substrates, namely, the peptidyl-tRNA and the incoming amino acyl-tRNA, it is proposed that this shift in the SecM-tRNA explains why peptide bond formation cannot occur and translation stalls.
doi:10.1371/journal.pbio.1000581
PMCID: PMC3022528  PMID: 21267063
22.  The SmpB C-terminal tail helps tmRNA to recognize and enter stalled ribosomes 
In bacteria, transfer-messenger RNA (tmRNA) and SmpB comprise the most common and effective system for rescuing stalled ribosomes. Ribosomes stall on mRNA transcripts lacking stop codons and are rescued as the defective mRNA is swapped for the tmRNA template in a process known as trans-translation. The tmRNA–SmpB complex is recruited to the ribosome independent of a codon–anticodon interaction. Given that the ribosome uses robust discriminatory mechanisms to select against non-cognate tRNAs during canonical decoding, it has been hard to explain how this can happen. Recent structural and biochemical studies show that SmpB licenses tmRNA entry through its interactions with the decoding center and mRNA channel. In particular, the C-terminal tail of SmpB promotes both EFTu activation and accommodation of tmRNA, the former through interactions with 16S rRNA nucleotide G530 and the latter through interactions with the mRNA channel downstream of the A site. Here we present a detailed model of the earliest steps in trans-translation, and in light of these mechanistic considerations, revisit the question of how tmRNA preferentially reacts with stalled, non-translating ribosomes.
doi:10.3389/fmicb.2014.00462
PMCID: PMC4151336  PMID: 25228900
SmpB; tmRNA; decoding; ribosome stalling; EFTu
23.  Cryo-EM visualization of the ribosome in termination complex with apo-RF3 and RF1 
eLife  2013;2:e00411.
Termination of messenger RNA translation in Bacteria and Archaea is initiated by release factors (RFs) 1 or 2 recognizing a stop codon in the ribosomal A site and releasing the peptide from the P-site transfer RNA. After release, RF-dissociation is facilitated by the G-protein RF3. Structures of ribosomal complexes with RF1 or RF2 alone or with RF3 alone—RF3 bound to a non-hydrolyzable GTP-analog—have been reported. Here, we present the cryo-EM structure of a post-termination ribosome containing both apo-RF3 and RF1. The conformation of RF3 is distinct from those of free RF3•GDP and ribosome-bound RF3•GDP(C/N)P. Furthermore, the conformation of RF1 differs from those observed in RF3-lacking ribosomal complexes. Our study provides structural keys to the mechanism of guanine nucleotide exchange on RF3 and to an L12-mediated ribosomal recruitment of RF3. In conjunction with previous observations, our data provide the foundation to structurally characterize the complete action cycle of the G-protein RF3.
DOI: http://dx.doi.org/10.7554/eLife.00411.001
eLife digest
Ribosomes are complex molecular machines that join amino acids together to form proteins. The order of amino acids in the protein is specified by a strand of messenger RNA (mRNA), and the process of decoding the mRNA into a string of amino acids is called translation. A ribosome consists of two subunits—one large, one small—that come together at a particular site on the mRNA strand called the translation initiation site. The ribosome then moves along the mRNA—joining together amino acids brought to it by transfer RNA (tRNA)—until it reaches a termination site and releases the protein.
The ribosome has three sites; the first amino acid to be delivered by a tRNA molecule to the ribosome occupies the site in the middle—also called the P site—and the second amino acid is delivered to the A site. Once the first two amino acids have been joined together, the ribosome moves along the mRNA so that the first amino acid now occupies the third site, called the E or exit site, and the second amino acid occupies the P site, leaving the A site vacant. The third amino acid is then delivered to the A site, and the whole process repeats itself until the ribosome reaches the termination site. Proteins called release factors are responsible for terminating the translation process and releasing the translated string of amino acids, which folds to form a protein. In bacteria this task can by performed by two releases factors, known as RF1 and RF2. However, the release factor must itself be released to leave the ribosome free to translate another strand of mRNA.
Pallesen et al. have used cryo-electron microscopy (cryo-EM) to study how a third release factor, RF3, helps to release RF1 from the ribosome in bacteria. In cells, RF3 usually forms a complex with a molecule called GDP, and the cryo-EM studies show that this molecule is released shortly after the RF3•GDP complex enters the ribosome. Once inside the ribosome, RF3 comes into contact with RF1 and with a protein called L12 that is part of the ribosome. A molecule called GTP—which is well known as a source of energy within cells—then binds to RF3, and this causes the shape of the ribosome to change. This change of shape results in the release of RF1 and the formation of a new RF3•GDP complex, which then leaves the ribosome.
Further work is needed to fully understand the role of L12 in these events, but a detailed understanding of the mechanism for terminating the translation of mRNA by the ribosome is coming into view.
DOI: http://dx.doi.org/10.7554/eLife.00411.002
doi:10.7554/eLife.00411
PMCID: PMC3677378  PMID: 23755360
Ribosome; Cryo-EM; Structure; RF1; RF3; L7/L12; E. coli
24.  Identification of residues required for stalled-ribosome rescue in the codon-independent release factor YaeJ 
Nucleic Acids Research  2013;42(5):3152-3163.
The YaeJ protein is a codon-independent release factor with peptidyl-tRNA hydrolysis (PTH) activity, and functions as a stalled-ribosome rescue factor in Escherichia coli. To identify residues required for YaeJ function, we performed mutational analysis for in vitro PTH activity towards rescue of ribosomes stalled on a non-stop mRNA, and for ribosome-binding efficiency. We focused on residues conserved among bacterial YaeJ proteins. Additionally, we determined the solution structure of the GGQ domain of YaeJ from E. coli using nuclear magnetic resonance spectroscopy. YaeJ and a human homolog, ICT1, had similar levels of PTH activity, despite various differences in sequence and structure. While no YaeJ-specific residues important for PTH activity occur in the structured GGQ domain, Arg118, Leu119, Lys122, Lys129 and Arg132 in the following C-terminal extension were required for PTH activity. All of these residues are completely conserved among bacteria. The equivalent residues were also found in the C-terminal extension of ICT1, allowing an appropriate sequence alignment between YaeJ and ICT1 proteins from various species. Single amino acid substitutions for each of these residues significantly decreased ribosome-binding efficiency. These biochemical findings provide clues to understanding how YaeJ enters the A-site of stalled ribosomes.
doi:10.1093/nar/gkt1280
PMCID: PMC3950681  PMID: 24322300
25.  Histidine 197 in Release Factor 1 is Essential for A Site Binding and Peptide Release 
Biochemistry  2010;49(43):9385-9390.
Class I peptide release factors 1 and 2 (RF1 and RF2) recognize the stop codons in the ribosomal decoding center and catalyze peptidyl-tRNA hydrolysis. High-fidelity stop codon recognition by these release factors is essential for accurate peptide synthesis and ribosome recycling. X-ray crystal structures of RF1 and RF2 bound to the ribosome have identified residues in the mRNA-protein interface that appear critical for stop codon recognition. Especially interesting is a conserved histidine in all bacterial class I release factors that forms a stacking interaction with the second base of the stop codon. Here we analyzed the functional significance of this conserved histidine (197 in E. coli) of RF1 by point mutagenesis to alanine. Equilibrium binding studies and transient-state kinetic analysis have shown that the histidine is essential for binding with high affinity to the ribosome. Furthermore, analysis of the binding data indicates a conformational change within the RF1•ribosome complex that results in a more tightly bound state. The rate of peptidyl-tRNA hydrolysis was also reduced significantly, more than the binding data would suggest, implying a defect in the orientation of the GGQ domain without the histidine residue.
doi:10.1021/bi1012047
PMCID: PMC2967428  PMID: 20873815
Ribosome; release factor; stop codon; translation; termination

Results 1-25 (715287)