Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press RNA (2001), 7:1708–1716+ Cambridge University Press+ Printed in the USA+ Copyright © 2001 RNA Society+ DOI: 10+1017+S1355838201011281 REPORT Increased sensitivity to protein synthesis inhibitors in cells lacking tmRNA JESÚS DE LA CRUZ1,2 and AGUSTÍN VIOQUE1 1 Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Américo Vespucio s/n, E-41092 Sevilla, Spain ABSTRACT tmRNA (also known as SsrA or 10Sa RNA) is involved in a trans-translation reaction that contributes to the recycling of stalled ribosomes at the 39 end of an mRNA lacking a stop codon or at an internal mRNA cluster of rare codons. Inactivation of the ssrA gene in most bacteria results in viable cells bearing subtle phenotypes, such as temperature- sensitive growth. Herein, we report on the functional characterization of the ssrA gene in the cyanobacterium Syn- echocystis sp. strain PCC6803. Deletion of the ssrA gene in Synechocystis resulted in viable cells with a growth rate identical to wild-type cells. However, null ssrA cells (DssrA) were not viable in the presence of the protein synthesis inhibitors chloramphenicol, lincomycin, spiramycin, tylosin, erythromycin, and spectinomycin at low doses that do not significantly affect the growth of wild-type cells. Sensitivity of DssrA cells similar to wild-type cells was observed with kasugamycin, fusidic acid, thiostrepton, and puromycin. Antibiotics unrelated to protein synthesis, such as ampicillin or rifampicin, had no differential effect on the DssrA strain. Furthermore, deletion of the ssrA gene is sufficient to impair global protein synthesis when chloramphenicol is added at sublethal concentrations for the wild-type strain. These results indicate that ribosomes stalled by some protein synthesis inhibitors can be recycled by tmRNA. In addition, this suggests that the first elongation cycle with tmRNA, which incorporates a noncoded alanine on the growing peptide chain, may have mechanistic differences with the normal elongation cycles that bypasses the block produced by these specific antibiotics. tmRNA inactivation could be an useful therapeutic target to increase the sensitivity of pathogenic bacteria against antibiotics. Keywords: antibiotics; ssrA; Synechocystis; tmRNA; translation INTRODUCTION cates that tmRNA plays a role in translation (for a re- view, see Muto et al+, 1998; Karzai et al+, 2000)+ tmRNA Bacterial cells contain a panoply of stable nontrans- can form in vivo a tRNA half molecule consisting of a lated RNAs such as ribosomal and transfer RNAs amino acid acceptor arm and a TCC stem-loop (Ko- (rRNAs and tRNAs; reviewed in Wassarman et al+, mine et al+, 1994)+ The presence of this tRNA-like struc- 1999)+ Amongst these RNAs, the so-called tmRNA (also ture is further supported by the fact that the precursor known as SsrA or 10Sa RNA) was one of the first to be of tmRNA (pre-tmRNA), as all pre-tRNAs, is 5 -end pro- identified in Escherichia coli (Jain et al+, 1982); how- 9 cessed by RNase P, and tmRNA is aminoacylated by ever, its function has remained elusive for a decade , alanyl-tRNA synthetase (Komine et al+, 1994)+ In addi- The tmRNA is encoded by the ssrA gene which is , highly conserved in all eubacteria (Knudsen et al+, 2001)+ tion the alanylated-tmRNA interacts with elongation fac- In vivo, tmRNA is tightly associated to the SmpB pro- tor Tu (EF-Tu) and GTP forming a ternary complex +, (Rudinger-Thirion et al+, 1999) and is found associ- tein and less tightly to other proteins (Karzai et al +, + , 1999; Karzai & Sauer, 2001)+ Different evidence indi- ated with 70S ribosomes (Tadaki et al 1996) Finally tmRNAs have a short reading frame coding for about 11 amino acids (Knudsen et al+, 2001), which are added Reprint requests to: Agustín Vioque, Instituto de Bioquímica Veg- etal y Fotosíntesis, Universidad de Sevilla-CSIC, Centro de Investi- to the C-terminus of an incomplete nascent protein trans- gaciones Científicas Isla de la Cartuja, Avda+ Américo Vespucio s/n, lated from truncated mRNAs (Keiler et al+, 1996)+ E-41092 Sevilla, Spain; e-mail: vioque@us+es+ Based on some of these observations, Sauer and 2 Present address: Departamento de Genética, Facultad de Bio- logía, Universidad de Sevilla, Reina Mercedes 6, E-41012 Sevilla, coworkers proposed the “trans-translation” model for Spain+ the biological function of tmRNA (Keiler et al+, 1996)+ 1708 Downloaded from rnajournal.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press tmRNA and translation inhibitors 1709 According to this model, alanine-charged tmRNA asso- tanabe et al+, 1998)+ For this reason, we undertook the ciated with EF-Tu enters the A site of a ribosome stalled functional characterization of the ssrA gene in the cy- at the 39 end of a truncated mRNA lacking a stop co- anobacterium Synechocystis sp+ strain PCC6803, in don+ Then, the noncoded alanine is incorporated to the the context of an ongoing project on the analysis of nascent polypeptide chain through a cycle of transla- stable RNAs from this cyanobacterium+ Herein, we re- tion elongation, before the mRNA-like domain of tmRNA port that Synechocystis cells disrupted for the ssrA gene replaces the truncated mRNA on the ribosome+ Then, (DssrA cells) show no growth defect phenotype under this domain is used as a template to add a short pep- normal laboratory conditions+ However, a DssrA strain tide to the nascent polypeptide before translation ter- is not viable when cultivated at low concentrations of minates and a tagged protein is released (for details, several protein synthesis inhibitors+ In addition, global see Keiler et al+, 1996; Muto et al+, 1998)+ The short protein synthesis is impaired at low concentration of peptide acts as a recognition sequence that directs the chloramphenicol in a DssrA strain but not in a wild-type protein to quick degradation by specific proteases strain+ We conclude that tmRNA has a role in recycling (Gottesman et al+, 1998; Levchenko et al+, 2000)+ There- stalled ribosomes when they are blocked by those pro- fore, tmRNA has a double function: First, ribosomes tein synthesis inhibitors+ We assume that the first elon- that are stalled at truncated mRNAs lacking a termina- gation cycle that incorporates the noncoded alanine to tion codon are released+ Second, the prematurely nascent polypeptide chains is resistant to the bound truncated proteins are rapidly degraded, avoiding ac- antibiotic+ cumulation+ Both function have been separated genet- ically by engineering cells that carry a ssrA gene coding RESULTS AND DISCUSSION for an altered tag that is resistant to proteases, or al- ternatively, that cannot be aminoacylated (Keiler et al+, tmRNA is dispensable for growth 1996; Withey & Friedman, 1999; Huang et al+, 2000)+ From these studies, it seems clear that ribosome re- To study the cellular function of Synechocystis tmRNA, lease and not protein tagging is the relevant function of we first disrupted the ssrA gene by replacing most of tmRNA (Keiler et al+, 1996; Withey & Friedman, 1999; the gene sequence with a kanamycin resistance (Kmr) Huang et al+, 2000)+ In addition, it has been demon- cassette (Fig+ 1A)+ Although the ssrA gene is not part of strated that tmRNA-mediated trans-translation occurs a polycistronic unit (Kaneko et al+, 1996), the cassette quite frequently in normally growing cells and not only was inserted in both orientations to control for hypo- at truncated mRNAs lacking a termination codon (Abo thetical polar effects+ The disrupted copy of ssrA could et al+, 2000)+ Tagging by tmRNA occurs at rare codons be completely segregated as tested by southern blot in an mRNA or even at the natural termination codon (data not shown)+ Therefore, cells lacking a functional (Roche & Sauer, 1999, 2001)+ This suggests that ribo- ssrA gene seem to be viable+ some pausing or stalling by a number of different causes To further confirm the nonessential role of tmRNA, can activate tmRNA function+ However, several ques- we determined its steady-state levels by northern blot tions remain unsolved: How does tmRNA recognize and primer extension analyses in wild-type and DssrA stalled ribosomes, what is the mechanism of incorpo- strains+ As seen in Figure 1B,C, disruption of the ssrA ration of the noncoded alanine, and how does the ribo- gene resulted in the loss of mature tmRNA+ In addition, some resume in-frame translation at the mRNA-like we detected an additional band in the wild-type strain domain of tmRNA? by primer extension analysis (Fig+ 1C, lane 1), which Moreover, despite its important biological function and we attribute to the precursor of tmRNA (Tous et al+, its ubiquitous presence in the eubacterial kingdom, the 2001)+ Altogether, these experiments indicate that ssrA precise physiological role of tmRNA still remains elu- is a dispensable gene in Synechocystis, in disagree- sive,as it is, in general, dispensable for cell viability and ment with the data from Sugita and coworkers for Syn- it has been proved to be essential only in Neisseria echococcus (Watanabe et al+, 1998)+ gonorrhoeae and Mycoplasma (Hutchison et al+, 1999; Huang et al+, 2000)+ However, cells lacking a functional Disruption of ssrA causes tmRNA exhibit a variety of subtle phenotypes such as no apparent growth defect slow growth at high temperature or under different stresses, reduced motility, inhibition of phage growth, To understand the nonessential role of tmRNA, the an increase in the activity