Current deal with: Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Reina Mercedes 6, E-41012 Sevilla, Spain.
Agustín Vioque, Jesús de la Cruz, Trans-translation and protein synthesis inhibitors, FEMS Microbiology Letters, Quantity 218, Difficulty 1, January 2003, Pages 9–14, https://doi.org/10.1111/j.1574-6968.2003.tb11491.x
Summary
1 Introduction
When ribosomes attain the three′ finish of an mRNA that lacks a termination codon they grow to be stalled. Bacterial cells have developed a posh mechanism, referred to as trans-translation, to deal with this drawback and recycle the stalled ribosomes. Trans-translation additionally comes into play in a number of different circumstances through which ribosomes grow to be stalled or paused for different causes. Trans-translation requires a secure RNA referred to as tmRNA (as a result of it capabilities each as switch and as messenger RNA; additionally referred to as 10Sa RNA or SsrA RNA) and a small fundamental protein that binds tmRNA referred to as SmpB (small protein B). tmRNA and SmpB are universally current in Micro organism however aren’t identified in Archaea and Eukaryotae, besides in chloroplasts from some algae and within the mitochondria of Reclinomonas americana[1].
There have been a number of latest evaluations on the mechanism of trans-translation and its organic function [2–5]. Right here, we summarize the present information on the totally different circumstances throughout ribosome elongation that may activate trans-translation. We additionally focus on the connection between trans-translation and protein synthesis inhibitors.
2 Mechanism and performance of trans-translation
tmRNA is encoded by the ssrA gene, which is very conserved in all eubacteria [1]. In vivo, tmRNA is tightly related to the SmpB protein and fewer tightly with different proteins [6,7]. A number of traces of proof point out that tmRNA performs a job in translation. tmRNA can in vivo kind a tRNA half molecule consisting of a amino acid acceptor arm and a TΨC stem-loop [8]. The presence of this tRNA-like construction is additional supported by the truth that the precursor of tmRNA, like all pre-tRNAs, is 5′ finish processed by RNase P and tmRNA is aminoacylated by alanyl-tRNA synthetase [8]. As well as, the alanylated tmRNA interacts with elongation issue Tu (EF-Tu) and GTP forming a ternary advanced [9] and is discovered related to 70S ribosomes [10]. SmpB protein is required for the interplay with the ribosome [6]. tmRNA has a brief studying body encoding about 11 amino acids, that are added to the C-terminus of an incomplete nascent protein translated from truncated mRNAs [11]. Primarily based on a few of these observations, Sauer and colleagues proposed the ‘trans-translation’ mannequin for the organic operate of tmRNA. In line with this mannequin, alanine-charged tmRNA related to EF-Tu and SmpB enters the empty A web site of a ribosome stalled on the 3′ finish of a truncated mRNA missing a cease codon. Then, the non-coded alanine is included into the nascent polypeptide chain by a cycle of translation elongation, earlier than the mRNA-like area of tmRNA replaces the truncated mRNA on the ribosome. Then, this area is used as a template so as to add a brief peptide to the nascent polypeptide earlier than translation terminates and a tagged protein is launched (for particulars see [2,11]). The quick tag acts as a recognition sequence that directs the protein to fast degradation by particular proteases [12,13]. Due to this fact, tmRNA has a double operate: first, ribosomes which are stalled at truncated mRNAs missing a termination codon are launched. Second, the prematurely truncated proteins are quickly degraded. Each capabilities have been separated genetically by engineering cells that carry a ssrA gene encoding an altered tag that isn’t inclined to proteolysis, or alternatively, that can not be aminoacylated [11,14,15]. From these research, it appears clear that ribosome launch and never protein tagging is the related operate of tmRNA [11,14,15]. As well as, it has been extra not too long ago demonstrated that tmRNA-mediated trans-translation happens fairly regularly in usually rising cells, and never solely at truncated mRNAs missing a termination codon [16] (Fig. 1). Tagging by tmRNA additionally happens at codons similar to low abundance tRNAs and even on the pure termination codon [17,18]. These observations recommend that ribosome pausing or stalling, both on the finish or inside the coding sequence of the mRNA, can activate tmRNA operate (Fig. 1). Nonetheless, a number of questions stay unsolved: how does tmRNA acknowledge paused or stalled ribosomes, what’s the mechanism of incorporation of the non-coded alanine, and the way does the ribosome resume in-frame translation on the mRNA-like area of tmRNA?
Generally, tmRNA isn’t important for cell viability underneath customary lab situations. ssrA or smpB null strains present no or delicate phenotypes, similar to slower progress, impaired restoration from carbon hunger [19], lowered progress at 45°C and motility on semisolid agar [8], elevated expression or exercise of the so-called Alp protease [20], enhanced sensitivity to excessive temperature of sure mutants [21], and inhibition of phage progress [6,22,23]. Thus far, the tmRNA has been proven to be important for progress solely in Neisseria gonorrhoeae[14]. It isn’t identified why tmRNA is important in N. gonorrhoeae however a number of speculative proposals have been superior [16]. Transposon mutagenesis means that tmRNA can also be important in Mycoplasma species [24]. As well as, tmRNA is required for full virulence of Salmonella[23].
3 Trans-translation and ribosome inhibitors
Not too long ago, we’ve discovered that ssrA null mutants of the cyanobacterium Synechocystis sp. PCC6803 are hypersensitive to a number of protein synthesis inhibitors [25]. This hypersensitive phenotype can also be noticed in Escherichia coli and Salmonella typhimurium ssrA null mutants [26] (A. Vioque, unpublished; see additionally Fig. 2) and in an E. coli smpB null mutant (A. Vioque, unpublished).
In our preliminary research, kanamycin couldn’t be examined as a result of the Synechocystis ssrA gene was disrupted with a kanamycin resistance cassette [25]. We’ve repeated these research with a S. typhimurium ssrA null pressure through which the ssrA gene was interrupted by a tetracycline cassette [23] and have discovered that the mutant pressure can also be hypersensitive to kanamycin (Fig. 2), as additionally described for E. coli[26]. Due to this fact, the hypersensitive phenotype appears to be a common characteristic of cells unable to trans-translate, by inactivation of both the tmRNA or the SmpB protein. As well as, we are able to conclude that trans-translation has a job in cell survival within the presence of sublethal concentrations for the wild-type cells of very various protein synthesis inhibitors.
Is it doable to conceive a single mechanism that explains the elevated resistance of cells with a purposeful trans-translation system to all of the number of protein synthesis inhibitors? Under we contemplate the totally different antibiotics in keeping with their mode of motion.
3.1 Antibiotics that induce misreading
The function of trans-translation has been studied in E. coli cells within the presence of aminoglycoside antibiotics that induce misreading [27], similar to kanamycin, gentamicin, hygromycin B, streptomycin, G418 and paromomycin [26]. As a consequence of misreading, these antibiotics additionally trigger translational read-through of cease codons and due to this fact extra ribosomes attain the three′ finish of regular mRNAs. On this state of affairs, trans-translation could also be concerned in dissociating the ribosome, equally to what occurs when ribosomes attain the three′ finish of a truncated mRNA missing cease codons. Due to this fact, the elevated sensitivity to those antibiotics of ssrA null mutants in contrast with wild-type cells could also be as a result of failure within the ssrA null cells of recycling the ribosomes which have stalled on the 3′ finish of regular mRNAs as a result of translational read-through. The truth that cells carrying a mutant tmRNA that encodes a protease-resistant tag have an antibiotic resistance just like that of the wild-type cells [26] means that the trans-translation system confers elevated antibiotic resistance by the ribosome rescue operate somewhat than by degradation of irregular proteins.
Within the case of different inhibitors, further mechanisms should be thought of. As no detailed research have been carried out, we are able to solely speculate primarily based on the information of the mechanism of motion on these antibiotics. In these circumstances, trans-translation would come into play earlier than the ribosome reaches the three′ finish of the mRNA. As above, trans-translation may additionally launch the stalled ribosomes, which resume regular translation utilizing the tag-coding area of tmRNA. The state of affairs can be just like the function of trans-translation when dissociating ribosomes at uncommon or cease codons. We assume that at sublethal concentrations of antibiotics, solely a fraction of the ribosomes (lower than one ribosome per mRNA engaged in translation) grow to be stalled on the mRNA, and due to this fact the likelihood that an antibiotic molecule binds once more as soon as the inhibited ribosome has been launched by trans-translation is low. This easy assumption additionally requires that the primary spherical of translation with tmRNA, which includes the non-encoded alanine, is insensitive to the presence of the antibiotic, which is displaced to permit steady translation on the tmRNA. It isn’t shocking that the resumption of translation on tmRNA can be extra proof against the antibiotic, taking into consideration the variations in measurement and construction between tRNA and tmRNA, which might suggest variations within the structural transitions that happen throughout an elongation cycle with a standard tRNA or when resuming translation on tmRNA.
3.2 Peptidyl transferase inhibitors
Chloramphenicol and lincomycin have been proven to have a detrimental impact on progress of a ssrA null mutant of Synechocystis[25]. Comparable results have been proven for the E. coli ssrA null mutant when cells are handled with chloramphenicol [26]. Within the presence of peptidyl transferase inhibitors, ribosomes grow to be stalled with the A web site occupied by an aminoacyl-tRNA. This appears to be inconsistent with the conventional operate of tmRNA, which requires that the ribosomal A web site is free. Usually, after lodging of the cognate aminoacyl-tRNA within the A web site and EF-Tu launch, peptide bond formation may be very fast and the ensuing peptidyl-tRNA is within the A/P hybrid state. Nonetheless, within the presence of a peptidyl transferase inhibitor, the aminoacyl-tRNA stays within the A web site and its spontaneous launch would make it possible to tmRNA to enter the A web site (Fig. 1E) and resume translation.
Not too long ago, it has been proven that chloramphenicol additionally causes translational inaccuracy in vivo [28]. This leads to elevated read-through of cease codons, due to this fact extra ribosomes would attain the three′ finish of the mRNA, equally to what occurs with aminoglycosides mentioned earlier.
3.3 Spectinomycin
From the a number of translocation inhibitors assayed, together with spectinomycin, fusidic acid and thiostrepton, solely spectinomycin confirmed a differential impact on the ssrA null mutants of Synechocystis. Spectinomycin inhibits an early step of translocation (EF-G·GTP binding) [29], due to this fact the ribosomal A web site can be occupied by the peptidyl-tRNA. On this state of affairs, it’s much more troublesome to clarify the mode of motion of tmRNA. Translocation can happen within the absence of EF-G at a gradual price [30]. Due to this fact, one can speculate that the tmRNA·SmpB·EF-Tu·GTP advanced can ‘push’ the peptidyl-tRNA to the P web site bypassing the requirement of EF-G for the primary translocation cycle within the trans-translation course of (Fig. 1F).
3.4 Macrolides
The ssrA null mutants of Synechocystis are additionally extra delicate than wild-type cells to erythromycin, spiramycin and tylosin. These antibiotics sterically block the peptide exit tunnel [31,32]. There are studies that conclude that erythromycin induces dissociation of peptidyl-tRNA [33]. If peptidyl-tRNA is launched from the ribosome, what can be the function of trans-translation? One provocative hypothesis is that ribosomes blocked by macrolides dissociate the peptidyl-tRNA and bind alanyl-tmRNA to provoke translation on the tag-coding sequence of tmRNA. On this respect, it is usually attention-grabbing to contemplate that in vitro, the alanyl-tmRNA from E. coli varieties a posh with alanyl-tRNA, alanine being the primary codon of the tmRNA inner open studying body (resume codon) [34]. This interplay could possibly be functionally related, facilitating the resumption of translation on the tag-coding sequence of tmRNA.
It’s believable to invest that the stress brought on by the presence of sublethal concentrations of protein synthesis inhibitors may enhance the requirement for tmRNA. On this state of affairs, no particular fashions can be required to clarify the hypersensitivity of ssrA mutants to those inhibitors. Certainly, in Bacillus subtilis, tmRNA is required for progress underneath a number of totally different stresses unrelated to protein synthesis [35]. Nonetheless, this appears to not be the case in Synechocystis, the place no variations have been detected between wild-type and ssrA null mutants underneath different totally different annoying situations than the presence of protein synthesis inhibitors [25]. As well as, ssrA null mutants have elevated sensitivity solely to a subset of all of the protein synthesis inhibitors examined, suggesting that particular mechanisms of trans-translation motion might be related.
4 Trans-translation as a therapeutic goal – “protein synthesis inhibitors”
Independently of the mechanism by which trans-translation contributes to survival within the presence of protein synthesis inhibitors, it’s clear that inactivation of trans-translation has a synergistic impact with protein synthesis inhibitors on selling inhibition of cell progress. This impact is extra important with aminoglycoside antibiotics that induce misreading, similar to kanamycin and streptomycin. Particular medication that inactivate trans-translation could possibly be developed and utilized in mixture with aminoglycosides as a more practical device in opposition to pathogenic micro organism. Furthermore, the efficient focus of the protein synthesis inhibitor might be lowered within the presence of a trans-translation inhibitor, decreasing negative effects on well being. The rational improvement of trans-translation inhibitors requires information of the construction of the macromolecules concerned. An necessary step ahead has been the latest publication of the answer construction of SmpB [36]. After all, inhibitors of trans-translation could possibly be straight helpful in opposition to these pathogens through which this mechanism is important, similar to N. gonorrhoeae[14] and sure Mycoplasma[24], or these micro organism through which tmRNA performs a job in pathogenesis [23].
5 Concluding remarks
Ribosome inhibitors could possibly be a great tool as a probe to grasp the mechanism of trans-translation simply as they’ve been extraordinarily helpful within the elucidation of the conventional protein synthesis course of. The elevated sensitivity to protein synthesis inhibitors in several bacterial cells faulty in trans-translation means that tmRNA could possibly be a pretty goal for the event of recent particular medication. Eukaryotes don’t use trans-translation, however somewhat an unrelated mechanism to cope with stalled ribosomes blocked on faulty mRNAs [37]. Furthermore, tmRNA isn’t identified in animal cells [1]. Due to this fact, a particular drug directed in opposition to trans-translation is anticipated to have low toxicity.
The potential therapeutic use of inhibitors of trans-translation might be prolonged to inhibitors of bacterial RNase P, as a result of RNase P processing is required for the biosynthesis of tmRNA [8]. Not too long ago a number of such inhibitors have been developed that work at low concentrations [38]. Due to this fact, protein synthesis inhibitors and RNase P inhibitors might also have a synergistic impact on selling pathogenic bacterial inhibition.
Acknowledgements
We apologize to our colleagues who weren’t cited on this minireview due to house limitations. We thank Robert Sauer and Michael Mahan for strains. We’re grateful to Venkat Gopalan for suggesting the usefulness of RNase P inhibitors to deplete cells of tmRNA. Analysis in A.V.’s lab is supported by the Ministerio de Ciencia y Tecnología (grant BMC2001-3789) and the Junta de Andalucía (CVI215).
“protein synthesis inhibitors”