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Mitochondrial translation appears to involve two stalled-ribosome rescue factors (srRFs). One srRF is an ICT1 protein from humans that rescues a "non-stop" type of mitochondrial ribosomes (mitoribosomes) stalled on mRNA lacking a stop codon, while the other, C12orf65, reportedly has functions that overlap with those of ICT1; however, its primary role remains unclear. We herein demonstrated that the Saccharomyces cerevisiae homolog of C12orf65, Pth3 (Rso55), preferentially rescued antibiotic-dependent stalled mitoribosomes, which appear to represent a "no-go" type of ribosomes stalled on intact mRNA. On media containing a non-fermentable carbon source, which requires mitochondrial gene expression, respiratory growth was impaired significantly more by the deletion of PTH3 than that of the ICT1 homolog PTH4 in the presence of antibiotics that inhibit mitochondrial translation, such as tetracyclines and macrolides. Additionally, the in organello labeling of mitochondrial translation products and quantification of mRNA levels by quantitative RT-PCR suggested that in the presence of tetracycline, the deletion of PTH3, but not PTH4, reduced the protein expression of all eight mtDNA-encoded genes at the post-transcriptional or translational level. These results indicate that Pth3 can function as a mitochondrial srRF specific for ribosomes stalled by antibiotics and plays a role in antibiotic resistance in fungi.

Ribosomes often stall during protein synthesis in various situations in a cell, either unexpectedly or in a programmed fashion. While some of them remain stalled for gene regulation, many are rescued by some cellular systems. Ribosomes stalled at the 3' end of a truncated mRNA lacking a stop codon (non-stop mRNA) are rescued by trans-translation mediated by tmRNA (transfer-messenger RNA) and a partner protein, SmpB. Through trans-translation, a degradation tag is added to the C-termini of truncated polypeptides from a truncated mRNA to prevent them from accumulation in the cell. Trans-translation has crucial roles in a wide variety of cellular events, especially under stressful conditions. The trans-translation system is thought to be universally present in the bacterial domain, although it is not necessarily essential in all bacterial cells. It has recently been revealed that two other systems, one involving a small protein, ArfA, with RF2 and the other involving YaeJ (ArfB), a class I release factor homologue, operate to relieve ribosome stalling in Escherichia coli. Thus, many bacterial species would have multiple systems to cope with various kinds of stalled translation events., (Copyright © 2014 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.)

Trans-translation is a bacterial quality control system in protein synthesis facilitated by transfer-messenger RNA (tmRNA). Here, we describe the in vitro system using purified factors to evaluate the two steps of trans-translation: peptidyl-transfer from peptidyl-tRNA to alanyl-tmRNA and decoding of the resume codon on tmRNA.

Ribosomes are trapped at the 3′ ends of mRNAs that lack a natural stop codon. In bacteria, a reaction called trans-translation recycles ribosomes entrapped at such ‘non-stop’ mRNAs. The main player in trans-translation is tmRNA (SsrA-RNA), a bi-functional RNA that acts as both a tRNA and an mRNA. In the trans-translation reaction, alanine-charged tmRNA loads at the ribosomal A-site and translation shifts to the resume codon in tmRNA. Translation of tmRNA stops at a natural stop codon at the end of the small reading frame of tmRNA. In this way, the reaction simultaneously adds a peptide tag to the end of the nascent, incomplete polypeptide and recycles the stalled ribosomes. The peptide tag is recognized by cellular proteases that rapidly degrade the incomplete, potentially harmful polypeptides. The trans-translation reaction is not essential in most bacteria, raising the possibility that ribosomes stalled at non-stop mRNAs can be rescued by alternative routes. In this issue of Molecular Microbiology, Chadani et al. show that a novel translation factor, ArfA, can recycle a ribosome trapped at the 3′ end of a non-stop mRNA in the absence of trans-translation. AfrA is essential in the absence of tmRNA, showing that the two systems work in parallel to resolve stalled ribosomes.

Trans-translation relieves a stalled translation on the bacterial ribosome by transfer-messenger RNA (tmRNA) with the help of SmpB, an essential cofactor of tmRNA. Here, we examined the role of the unstructured C-terminal tail of SmpB using an in vitro trans-translation system. It was found that truncation of the C-terminal tail or substitution of tryptophan residue at 147 in the middle of the C-terminal tail affected the activity in the early stage of trans-translation. Our investigations also revealed that the C-terminal tail is not required for the events until GTP is hydrolyzed by EF-Tu in complex with tmRNA-SmpB. A synthetic peptide corresponding to the C-terminal tail of SmpB inhibited peptidyl-transfer of alanyl-tmRNA and A-site binding of SmpB, but not GTP hydrolysis. These results suggest that the C-terminal tail has a role in the step of accommodation of alanyl-tmRNA-SmpB into the A-site. Directed hydroxyl radical probing indicated that tryptophan residue at 147 is located just downstream of the decoding center in the mRNA path when SmpB is in the A-site.

tmRNA has a dual function both as a tRNA and an mRNA to facilitate trans-translation, in which a ribosome can switch translation from a truncated or other problematic mRNA to the tmRNA's tag-encoding sequence. During trans-translation, tmRNA enters the ribosomal A-site without a codon-anticodon interaction, but with an SmpB which is a tmRNA binding protein. To further study the interaction of SmpB with ribosome and tmRNA, we have performed directed hydroxyl radical probing. It revealed that there are two SmpB binding sites at the A-site and P-site, and their C-terminal regions reside along the mRNA path in the 30S subunit. From these results, we propose a new type of molecular mimicry, in which both tmRNA and SmpB mimic the structures and functions of tRNA and mRNA during trans-translation, addressing how tmRNA preferentially recognizes the stalled ribosome, and what substitutes for a codon-anticodon interaction.

Summary: Spore formation in Bacillus subtilis is significantly impaired by the deletion of the gene for tmRNA (ssrA), which facilitates the trans-translation reaction that rescues stalled ribosomes and degrades incompletely synthesized peptides. Microscopic analysis revealed that the sporulation of most DeltassrA cells is blocked after forespore formation. Expression analysis of lacZ-fused genes directed by several RNA polymerase sigma factors showed that the synthesis of active sigma(K), encoded by the sigK gene, is predominantly inhibited in DeltassrA cells. The defect in sigma(K) synthesis is attributable to a defect in the skin element excision, which generates the sigK gene, caused in turn by reduced expression of SpoIVCA (recombinase) in DeltassrA cells.

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.

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.

tmRNA has a dual function as a tRNA and an mRNA to relieve the stalled ribosome. During this process, tmRNA enters the ribosomal A-site without a codonanticodon interaction, but with a protein factor SmpB. Here, we established in vitro trans-translation system, which is able to evaluate peptidyl transfer to alanyltmRNA and translation of the resume codon on tmRNA. Using this system, the effects of mutations on tmRNA or SmpB are assessed by measuring the incorporations of labeled amino acid into polypeptide. Interaction of SmpB with ribosome and tmRNA was studied by directed hydroxyl radical probing. Our results revealed that SmpB constitutes an important element during trans-translation. We propose a new model of transtranslation.

In trans-translation, ribosome switches the template for protein synthesis from an mRNA to tmRNA. The molecular mechanism of trans-translation remains mysterious. In order to clarify how tmRNA move through the ribosome, we developed in vitro systems to monitor trans-translation as well as translation, which are composed of ribosome, elongation factors, tmRNA and SmpB all from Thermus thermophilus. In these systems, the early steps of trans-translation including trans-transfer and the resume codon decoding could be monitored. Using these systems, the function of ribosomal protein S1 which has been suggested to be involved in trans-translation was investigated.

Trans-translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of several kinds of aminoglycoside antibiotics, on trans-translation in vitro. A chemical footprinting study revealed that paromomycins bind tmRNA in the tRNA domain and in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound in the tRNA domain inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA binding protein. It was also found that paromomycin causes a shift of the translation-resuming point on tmRNA by -1. The effect on initiation-shift was suppressed by a mutation at the paromomycin binding site in 16S rRNA, but not by mutations in tmRNA. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome. A minimal structure that causes initiation-shift was identified.

trans-Translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. The results of a chemical footprinting study indicated that paromomycin molecules bind tmRNA at G332/G333 in the tRNA domain and A316 in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound at G332/G333 inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA-binding protein. It was also found that paromomycin causes a shift of the translation resuming point on tmRNA by -1. The effect on initiation shift was canceled by a mutation at the paromomycin-binding site in 16 S rRNA but not by mutations in tmRNA. A high concentration of paromomycin inhibited trans-translation, whereas it enhanced the initiation-shifted trans-translation when SmpB was exogenously added or a mutation was introduced at 333. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.

Trans-translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. A chemical footprinting study revealed that paromomycins bind tmRNA in the tRNA domain and in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound in the tRNA domain inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA binding protein. It was also found that paromomycin causes a shift of the translation-resuming point on tmRNA by -1. The effect on initiation-shift was canceled by a mutation at the paromomycin binding site in 16S rRNA, but not by mutations in tmRNA. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.

tmRNA has a dual function both as tRNA and mRNA and facilitates trans-translation. We observed the tagging derived from tmRNA from Bacillus subtilis in vivo and in vitro. Our studies give progressive suggestions on the mechanism of trans-translation.

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

tmRNA facilitates a novel translation, trans-translation, in which a ribosome can switch between translation of a truncated mRNA and the tmRNA's tag sequence. The mechanism underlying resumption of translation at a definite position is not known. In the present study, the effects of mutations around the initiation point of the tag-encoding sequence of Escherichia coli tmRNA on the efficiency and the frame of tag translation were assessed by measuring the incorporations of several amino acids into in vitro poly (U)-dependent tag-peptide synthesis. One-nucleotide insertions within the tag-encoding region did not shift the frame of tag translation. Any 1-nt deletion within the span of -5 to -1, but not at -6, made the frame of tag translation heterologous. Positions at which a single base substitution caused a decrease of trans-translation efficiency were concentrated within the span of -4 to -2. In particular, an A-4 to C-4 mutation seriously damaged the trans-translation, although this mutant retained normal aminoacylation and ribosome-binding abilities. A possible stem and loop structure around this region was not required for transtranslation. It was concluded that the tag translation requires the primary sequence encompassing -6 to +11, in which the central 3 nt, A-4, G-3, and U-2, play an essential role. It was also found that several base substitutions within the span of -6 to -1 extensively shifted the tag-initiation point by -1.

Transfer-messenger RNA (tmRNA) is a unique molecule that combines properties from both tRNA and mRNA, and facilitates a novel translation reaction termed trans -translation. According to phylogenetic sequence analysis among various bacteria and chemical probing analysis, the secondary structure of the 350-400 nt RNA is commonly characterized by a tRNA-like structure, and four pseudoknots with different sizes. A mutational analysis using a number of Escherichia coli tmRNA variants as well as a chemical probing analysis has recently demonstrated not only the presence of the smallest pseudoknot, PK1, upstream of the internal coding region, but also its direct implication in trans -translation. Here, NMR methods were used to investigate the structure of the 31 nt pseudoknot PK1 and its 11 mutants in which nucleotide substitutions are introduced into each of two stems or the linking loops. NMR results provide evidence that the PK1 RNA is folded into a pseudoknot structure in the presence of Mg(2+). Imino proton resonances were observed consistent with formation of two helical stem regions and these stems stacked to each other as often seen in pseudoknot structures, in spite of the existence of three intervening nucleo-tides, loop 3, between the stems. Structural instability of the pseudoknot structure, even in the presence of Mg(2+), was found in the PK1 mutants except in the loop 3 mutants which still maintained the pseudoknot folding. These results together with their biological activities indicate that trans -translation requires the pseudoknot structure stabilized by Mg(2+)and specific residues G61 and G62 in loop 3.

10Sa RNA is a bacterial small stable RNA, in which the 5' and 3'-terminal sequences can be folded into a tRNA-like secondary structure which can be aminoacylated with alanine. It was found that Escherichia coli 10Sa RNA facilitated the incorporation of alanine, tyrosine, aspartic acid and glutamic acid, but not valine, isoleucine, serine or arginine, into the growing polypeptide in vitro, depending on poly (U)-directed poly-phenylalanine synthesis. This result indicates that 10Sa RNA functions as an mRNA for the tag-peptide which has been found to be attached to the C termini of truncated polypeptides synthesized in vivo. Aminoacylation with alanine was required for tag-specific amino acid incorporation and for efficient association of 10Sa RNA with the ribosome, indicating that 10Sa RNA also functions as an alanine tRNA in the tag-peptide synthesis. The dual function of 10Sa RNA both as an mRNA and as a tRNA in vitro strongly supports the trans translation hypothesis.

Here we show that Escherichia coli tmRNA (10Sa RNA) has a dual function both as an mRNA and as a tRNA in vitro. The function as a tRNA is prerequisite for the function as an mRNA. These observations strongly support the trans-translation hypothesis.

10Sa RNA is a small stable bacterial RNA in which the 5'- and 3'-end sequences are folded into a tRNA-like structure. The RNA is aminoacylatable with alanine in vitro, and it interacts with 70S ribosomes in the cell. Recently, Escherichia coli 10Sa RNA has been shown to contain the sequence-encoding tag-peptides, which are found to attach to the C-termini of truncated peptides synthesized in vivo. We have found that the E coli 10Sa RNA stimulates incorporation of the tag-specific amino acids into proteins depending on the poly(U)-directed poly-phenylalanine synthesis in the in vitro translation system. Our finding supports the 'trans-translation' model proposed for the tag synthesis, in which alanyl-10Sa RNA enters the ribosome when translation stops at the 3'-end of the truncated mRNA lacking a stop codon, and translation of the tag-peptide occurs by switching the template from mRNA to 10Sa RNA. In this unique reaction, 10Sa RNA acts both as a tRNA and as an mRNA.