Enteric Virulence Associated Protein Vapc Inhibits Translation by Cleavage of Initiator Trna

Enteric virulence associated protein VapC inhibits translation by cleavage of initiator tRNA

Kristoffer S. Winther and Kenn Gerdes1

Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle NE2 4HH, United Kingdom

Edited* by Susan Gottesman, National Cancer Institute, Bethesda, MD, and approved March 22, 2011 (received for review December 29, 2010)

Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases Typhimurium LT2, encoded by bona fide vapBC loci, are site- involved in quality control, metabolism and maturation of mRNA specific tRNases that cleave initiator tRNA between the antico- and rRNA. The majority of prokaryotic PIN-domain proteins are don stem and loop. encoded by the abundant vapBC toxin—antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric Results VapCs are site-specific endonucleases that cleave tRNAfMet in the VapC Inhibits Translation In Vitro. We showed previously that over- anticodon stem-loop between nucleotides þ38 and þ39 in vivo production of VapC inhibits global cellular translation (21). To and in vitro. Consistently, VapC inhibited translation in vivo and identify VapC’s target within the translational machinery, we pur- in vitro. Translation-reactions could be reactivated by the addition ified VapC and VapCLT2. Addition of purified, native VapC to an of VapB and extra charged tRNAfMet. Similarly, ectopic production in vitro translation reaction abolished translation (Fig. 1A, lanes 1 of tRNAfMet counteracted VapC in vivo. Thus, tRNAfMet is the only and 2). Preincubation with VapB antitoxin neutralized VapC cellular target of VapC. Depletion of tRNAfMet by vapC induction activity, showing that VapCs inhibition of translation was specific was bacteriostatic and stimulated ectopic translation initiation (lane 3). Thus, the preparation of VapC was active in vitro and its at elongator codons. Moreover, addition of chloramphenicol to activity could be counteracted by VapB. For reasons unknown, cells carrying vapBC induced VapC activity. Thus, by cleavage of VapCLT2 was not active in our in vitro assays and was therefore tRNAfMet, VapC simultaneously may regulate global cellular trans- analyzed in vivo only. lation and reprogram translation initiation. VapC Inhibits Translation by Degradation of Initiator tRNAfMet. Ectopic toxin-antitoxin ∣ tRNase ∣ riboendonuclease ∣ tRNA ∣ RNase production of VapC in vivo did not affect the degradation- patterns of lpp, dksA,orompA mRNAs, ribosomal RNAs, or rokaryotic toxin—antitoxin (TA) loci code for two compo- tmRNA (21). Consistently, VapC exhibited only weak RNase Pnents, a toxin that inhibits cell growth and an antitoxin that activity towards MS2 RNA in vitro (Fig. S2). Moreover, VapC counteracts the toxin. In Type I TA loci, the antitoxins are small did not associate specifically with 70S, 50S, or 30S ribosomal sub- antisense RNAs that repress translation of the toxin genes (1, 2) units (Fig. S3). Therefore, we considered that VapC might target while in Type II loci, the antitoxins are proteins that combine with tRNAs. VapC completely degraded purified E. coli tRNAfMet in a and neutralize the toxins (3). Type III TA loci encode small RNAs 15′ reaction (Fig. 1C). In contrast, tRNAVal and tRNAPhe were that combine with and neutralize the toxins (4). Based on toxin not degraded. Cleavage of tRNAfMet was counteracted by the sequence similarities, Type II loci have been divided into gene prior addition of VapB to VapC, again showing specificity. EDTA families (3, 5). Many of these gene families are present in both also inhibited the reaction, revealing that the reaction required bacteria and archaea, predicting that the cellular targets of the divalent cations (Fig. 1C). The in vitro translation reaction could toxins are of a general nature. Consistently, the RelE family of only be reactivated by the addition of both VapB and fMet- toxins, which is present in both prokaryotic domains, cleave tRNAfMet (Fig. 1B). The ability of fresh fMet-tRNAfMet to reac- mRNAs at codons positioned at the ribosomal A-site (6, 7). RelE tivate the reaction after quenching of VapC activity (compare toxins from archaea cleave mRNAs at A-site codons in Escher- lanes 3 and 4), shows that VapC does not have other targets with- ichia coli (8) and RelE from E. coli cleave A-site codons of mam- in the translational machinery. That tRNAfMet is the sole cellular malian and mitochondrial ribosomes (9, 10). The most abundant target of VapC was further substantiated by the observation that Type II TA loci are vapBC that encode PIN-domain toxins. Inter- ectopic overexpression of tRNAfMet counteracted the toxic effect estingly vapBC are highly abundant in some organisms. For of a vapC pulse (Fig. S4). example, the major human pathogen Mycobacterium tuberculosis and the hyperthermophilic chrenarchaeote Sulfolobus solfataricus VapC Cleaves Initiator tRNA Between the Anticodon Stem and Loop. have at least 45 and 30 vapBC loci, respectively (11, 12). PIN-do- VapC degraded full-length tRNAfMet into smaller fragment(s) mains (PilT N-terminal) consist of approximately 140 amino acids (Fig. 1C), indicating endonucleolytic cleavage. The cleavage was characterized by a quartet of conserved, negatively charged ami- mapped to occur between þ38 and þ39, at the anticodon stem- no acid residues configured in an RNase H-like fold (Fig. S1) loop boundary (Fig. S5). Thus, VapC is a site-specific tRNAfMet (13). In eukaryotes, PIN-domain proteins function in several endo-nuclease in vitro. The 3′-terminus of the 5′-product of types of RNA metabolism, such as nonsense-mediated mRNA tRNAfMet could not be ligated to [5′-32P]Cytidine 3’,5’-bispho- decay (14, 15), rRNA maturation (16), and mRNA turnover sphate (pCp) with T4 RNA ligase, consistent with a 2′,3′-cyclic (17–19). Ectopic production of VapC from both Gram positive and negative bacteria efficiently inhibits cell growth by inhibiting the global rate of translation (11, 20, 21). As with other Type II Author contributions: K.S.W. and K.G. designed research; K.S.W. performed research; TA loci, VapB antitoxins neutralize cognate VapCs by direct pro- K.S.W. and K.G. analyzed data; and K.S.W. and K.G. wrote the paper. tein—protein interaction (21). Even though several studies The authors declare no conflict of interest. showed that VapC has nonspecific ribo- or deoxyribonuclease *This Direct Submission article had a prearranged editor. activity in vitro, the specific target of VapC within the translation Freely available online through the PNAS open access option. apparatus has remained elusive (11, 13, 21, 22). Here we show 1To whom correspondence should be addressed. E-mail: [email protected]. that VapC (MvpT) of Shigella flexneri 2a virulence plasmid This article contains supporting information online at www.pnas.org/lookup/suppl/ BIOCHEMISTRY pMYSH6000 (23) and VapCLT2 of Salmonella enterica serovar doi:10.1073/pnas.1019587108/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1019587108 PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 ∣ 7403–7407 Downloaded by guest on September 26, 2021 Thus, VapC cleavage of tRNAfMet was highly specific. It should be noted that tRNAfMet isoforms of E. coli, S. flexneri, and S. enterica are identical, validating the use of E. coli as the host organism in these experiments. To confirm this inference, we fMet showed that tRNA of S. enterica was also cleaved after vapCLT2 induction (Fig. S7). The complete cleavage seen both in vitro and in vivo indicates that VapC can cleave both charged and uncharged tRNAfMet (Figs. 1C and 2A). The in vivo VapC cleavage site was mapped by isolating tRNA fragments using a biotinylated tRNAfMet-specific probe. Cells fMet expressing VapC or VapCLT2 produced tRNA -derived RNAs that had sizes identical to those obtained in vitro (Fig. 2B). The slightly smaller RNA fragments also observed in vivo most likely reflect nibbling of the RNA ends after the initial cleavage. Again, neither chloramphenicol nor induction of relE produced detectable cleavage products in vivo (Fig. 2B). A sequence alignment of tRNAfMet and tRNAMet (Fig. 2C) showed that cleavage occurred between the anticodon loop and the tRNAfMet specific GC nucleotide basepair stem that acts as a discriminator for tRNAfMet loading into the ribosomal P-site (25). The GC stem is conserved in initiator tRNAs in all three domains of life.

Change of a Predicted Catalytic, Highly Conserved aa Residue Abolishes VapC Activity. The catalytic centers of PIN-domain proteins consist of quartets of conserved, acidic aa residues (13). The sequence alignment in Fig. S1 predicted that the conserved aspartate at þ7 of VapC is required for catalytic activity. To test this possibility, we changed the aspartate to alanine. As described in the following, VapCD7A indeed was inactive in vivo.

Depletion of tRNAfMet by VapC Is Reversible and Bacteriostatic. It has been debated whether toxins encoded by TA loci are bacteriocidal or bacteriostatic. We followed the cellular content of fMet Fig. 1. VapC inhibits translation in vitro by cleavage of tRNAfMet.(A) Native tRNA in a physiological growth experiment. After induction VapC inhibits translation in vitro and can be neutralized by VapB. Each reac- of vapC or vapCLT2, cell-growth ceased rapidly (Fig. 3A). μ μ μ D7A tion contained the following: 6 L Premix, 4.5 L S30 Extract, 1.5 L amino In contrast, induction of vapCLT2 , that encodes a predicted acid mix without methionine, 0.5 μL 35S-methionine, and 4.5 pmol VapC with catalytically inactive VapC, did not inhibit cell growth. Thirty min- and without preincubation with 30 pmol VapB. The reaction was incubated utes later, vapC expression was terminated and cognate vapB μ ∼0 7 for 5 min at 37 °C before addition of 1 L MS2 RNA ( . pmol) (Roche). The genes were induced. As seen, cell growth resumed ≈30 min after reactions were incubated for 1 h at 37 °C before termination of the reaction ≈90 by acetone precipitation. The protein products were visualized by SDS-PAGE vapBLT2 induction and min after vapB induction. The slower and phosphorimaging. (B) Inhibition of translation in vitro by VapC can be recovery after vapB induction was correlated with a more reversed by fMet-tRNAfMet. VapC (9 pmol) (lanes 2, 3, and 4) or buffer (lane complete depletion and slower return of the cellular content fMet 1) were added to an E. coli S30 extract and incubated for 5′ before VapB of the tRNA (Fig. 3 B and C). These results show that VapC (60 pmol) (lane 3 and 4) or buffer (lane 1 and 2) was added at þ50 and can control the growth rate by depletion of tRNAfMet. The rapid incubation was continued; at þ100, fMet-tRNAfMet (lane 3) or buffer (lane resumption of cell growth and tRNAfMet recovery after induction 35 1, 2, and 4) was added together with S-met and MS2 RNA. The reaction of vapB is consistent with our previous conclusion that ectopic was terminated after incubation for an additional 10′.(C) VapC cleaves fMet fMet expression of VapC is bacteriostatic rather than bacterioci- tRNA . VapC (2.5 pmol or 5 pmol) was incubated with 2 pmol tRNA , dal (21). tRNAVal or tRNAPhe for 15′. Controls: VapC (5 pmol) was preincubated with either VapB (30 pmol) or 12.5 mM EDTA before the addition of tRNAfMet. We showed previously that abrupt reductions in growth rate, (D) Primary and secondary structures of E. coli fMet-tRNAfMet. such as the addition of chloramphenicol to rapidly growing cells, strongly activated transcription of the vapBC operon (21). This observation, however, did not tell if VapC was activated or not. phosphate. The 5′-terminus of the 3′-product could be phos- The knowledge of the VapC target, however, has provided a very phorylated without prior dephosphorylation, indicating a 5′-hy- sensitive assay of VapC activity. As seen from Fig. 3D, addition of droxyl group. These observations suggest that VapC generates chloramphenicol to cells carrying vapBC on a low-copy-number products similar to those of tRNA splicing endonucleases and plasmid induced detectable cleavage of tRNAfMet. No such clea- self-cleaving ribozymes (24). Classical RNases, such as RNase vage was seen when the plasmid carried vapBCD7A. Thus, reduc- T1 and A also generate these termini, but proceed to hydrolyse tion of the growth rate can induce VapC activity, consistent with the cyclic phosphodiester intermediate. decay of VapB under this condition. The tRNase activity of VapC was investigated in E. coli. VapCLT2 from S. enterica was included in this analysis. Northern VapC Activates Initiation of Translation at Elongator Codons. RelE is blotting showed that induction of vapC or vapCLT2 led to a rapid a ribosome-dependent RNase that cleaves mRNA at A-site decrease in full-length tRNAfMet, with a concomitant appearance codons, between the second and third base (6, 7). Consequently, of smaller cleavage products (Fig. 2A top). Neither chloramphe- RelE and its homologues can be used to map the position of nicol nor induction of relE had this effect. Importantly, induc- ribosomes on a given mRNA with high resolution using primer Met tion of vapC or vapCLT2 did not lead to cleavage of tRNA or extension analysis (10). Inhibition of translation by vapC or Arg His Phe seven other elongator tRNAs (tRNA 2, tRNA , tRNA , vapCLT2 induction leads to activation of YoeB, an E. coli RelE Thr Tyr Val Leu tRNA 1, tRNA , tRNA , or tRNA ) (Fig. 2A, Fig. S6). homologue that also cleaves mRNA between the second and

7404 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1019587108 Winther and Gerdes Downloaded by guest on September 26, 2021 Fig. 2. VapC cleaves tRNAfMet in vivo. (A) Northern blotting analysis of tRNAfMet (upper) and tRNAMet (lower). MG1655Δlon containing pKW3352HC

(pBAD33 ∷ vapCLT2−H6), pKW3382HC (pBAD33 ∷vapC−H6), or pKP3035 (pBAD33∷relE) were grown exponentially in LB medium at 37 °C. At time zero, arabinose (0.2%) was added to induce transcription. Samples were taken at time points indicated (min). Chloramphenicol (50 μg∕mL), Cm, was added to MG1655Δlon as a control. The use of a Δlon strain prevented fortuitous induction of mRNases by VapC overexpression (21, 26). (B) Analysis of the tRNAfMet cleavage product in vivo. tRNAfMet was isolated from total tRNA samples taken in (A)at10′ by hybridization to a biotinylated probe complementary to the 5′-cleavage product of tRNAfMet (Fmet-bio). The RNA/DNA hybrid was subsequently purified using Streptavidin sepharose and labeled with 32P in its 5′-end. M is untreated tRNAfMet and lane C is in vitro cleavage of tRNAfMet by VapC. FL: Full-length tRNAfMet; CL: Cleavage product of tRNAfMet.(C) Alignment of methionine accepting tRNAs of E. coli K-12.

third bases of A-site codons (21, 26). We showed previously that lacking initiation of translation at the AAA and AAG elongator

vapC induction activated YoeB such that YoeB cleaved dksA codons. Strikingly, induction of vapCLT2 resulted in ≈20-fold mRNA within its stop codon (26). This cleavage required trans- (AAA) and ≈4-fold (AAG) increases in the LucF/LucR ratios. lation of dksA mRNA. Here, we used vapC-induced YoeB- No increase was observed when translation was inhibited with dependent mRNA cleavage as a sensitive measure of mRNA chloramphenicol or yoeB induction. These results confirmed translation. We constructed three variants of dksA mRNA in that vapC induction activated ectopic translation initiation at which the Shine and Dalgarno (SD) sequence, the start-codon, elongator codons. or both were changed (Fig. 4A). Consistent with previous results, vapC induction resulted in ribosome-dependent YoeB cleavage Discussion within the stop codon of wild-type dksA mRNA (denoted SDwt We show here that enteric VapCs are RNases that cleave initiator AUG mRNA in Fig. 4A) (Fig. 4B, left, lanes 1, 2, and 5). This tRNA between the anticodon stem and loop. Depletion of cleavage disappeared if the yefM yoeB TA locus was deleted from tRNAfMet inhibited translation and, unexpectedly, activated the chromosome (lanes 4 and 7) showing that the cleavage initiation of translation at elongator codons positioned correctly depended on activation of YoeB. Consistently, induction of yoeB relative to a ribosome binding site. Even though other site- encoded by a plasmid led to efficient cleavage of the wild-type specific tRNases are known, namely colicin D, colicin E, and dksA mRNA (lane 8). Mutational change of the SD-sequence PrrC, VapC is so far the only tRNase known that cleaves initiator abolished stop codon cleavage in all three cases, showing that tRNA. Colicin D cleaves four isoaccepting arginine tRNAs YoeB cleavage required that the mRNA was translated (lanes between þ38 and þ39 (27), while colicin E5 cleaves in the antic- 3, 6, and 9). Next, we performed a similar series of experiments odon loop of four different elongator tRNAs, between þ34 and on mRNAs in which the AUG start-codon had been changed to þ35 (28). Colicins are considered to be bacteriocidal, and, in con- an elongator codon, AAG (Fig. 4B, right). Induction of yoeB did trast to VapC, are transported out of the producer cells to kill not lead to cleavage of the SD AAG mRNA, showing that the wt nonproducing competitor cells. Colicin-producing cells also reading-frame starting with AAG was normally not translated produce immunity proteins that are analogous in function to (lanes 15, 16). In contrast, the SD AAG mRNA was cleaved wt antitoxins. PrrC, which cleaves lysine tRNA in the anticodon after vapC induction (lanes 11 and 13). VapC-induced cleavage loop between þ34 and þ35, is activated by bacteriophage T4 of the SDwt AAG mRNA was abolished by mutational change of the SD sequence (lanes 12 and 14). This result shows that infection (29). VapC inhibited initiation of translation and simultaneously VapC and VapCLT2 induced translation at the AAG codon of dksA mRNA if the codon was positioned correctly relative to activated translation of reading frames that were normally silent. an SD sequence. Thus, induction of vapC activated initiation Whether this latter effect has physiological consequences (i.e., of translation at elongator codons. when endogenous VapC is activated in the wild-type context) To investigate this conjecture in a simpler and more general remains to be determined. However, Varshney and colleagues context, we used a dual luciferase assay to measure the fidelity observed that a reduction of the cellular level of tRNAfMet by of translation initiation. Firefly luciferase gene (lucF) had its mutations in the metZWV promoter relaxed the stringency of AUG start-codon changed to either AAA or AAG lysine codons; initiator tRNA selection at the ribosomal P-site (30). This result renilla luciferase (lucR) was included to normalize LucF activity supports that the reduced level of tRNAfMet seen after vapC

(Fig. 4C). The LucF/LucR ratios were very low for the AAA and induction allows loading of elongator tRNAs in the P-site and BIOCHEMISTRY

AAG constructs before induction of vapCLT2, consistent with the thereby stimulates ectopic initiation of translation. The require-

Winther and Gerdes PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 ∣ 7405 Downloaded by guest on September 26, 2021 Fig. 3. Correlation between vapC induction, cell growth, and tRNAfMet levels. (A) Bacterial growth after VapC expression and subsequent VapB

expression. Strains MG1655Δlon / pKW3352HC (pBAD33 ∷vapCLT2−H6)/

pKW51 (R1 ∷ pA1∕O4∕O3 ∷vapBLT2) (filled black diamond), MG1655Δlon / pKW3382HC (pBAD33 ∷vapC−H6) / pKW81 (pA1/O4/O3∷vapB) (filled black D7A square), or MG1655Δlon / pKW3353 (pBAD33 ∷vapCLT2−H6 ) (filled black tri- angle) were grown exponentially in LB medium at 37 °C. At 100′, vapC transcription was induced by the addition of 0.2% arabinose. In the case of strains

carrying vapC and vapCLT2, vapC expression was terminated at 130′ by the vapB addition of 0.4% glucose and transcription of and vapBLT2 was induced by 2 mM IPTG. (B) Northern blotting analysis on tRNAfMet of cell samples from A C fMet Fig. 4. Ectopic production of VapC induces initiation of translation by elon- ( ) Top: VapCLT2; Bottom: VapC ( ) Northern blotting analysis on tRNA fMet A B VapC 2 D7A. (D) Cleavage of tRNA by VapC activated from vapBC operon gator tRNAs. ( ) Drawing showing the four mRNAs analyzed in ( ). The top LT dksA during translation inhibition. MG1655/pKW254812 (vapBC) and MG1655/ wavy lines symbolize wild-type mRNA with SD, AUG start-codon, and D7A dksA pKW254813 (vapBC ) were grown exponentially in LB medium at 37 °C. UAA stop codon. SDmut indicates that the SD sequence of AGGAG, At time zero, 50 μg∕mL of chloramphenicol was added. Samples were taken was changed to UCCUC. In the two bottom mRNAs, an AAG lysine codon at time points indicated (min). The lane marked C shows tRNAfMet cleaved by replaces the AUG start codon. Arrows pointing at the UAA codons indicate dksA B VapC in vivo. CL denotes the VapC cleavage product(s) of tRNAfMet. The use of possible YoeB cleavage in the stop codon of .() Primer extension a Δlon strain in (A), (B), and (C) prevented ectopic induction of endogenous analysis of VapC-mediated ribosome-dependent YoeB cleavage. MG1655 or Δ yefM yoeB mRNases of E. coli (RelE, MazF, etc) by VapC overexpression (21, 26). MG1655 ( ) containing either pKW25420T (SDwt AUG), pKW25421T (SDwt AAG), pKW25427T (SDmut AUG), or pKW25428T (SDmut AAG) together

with pKW3352HC (pBAD33::vapCLT2-H6), pKW3382HC (pBAD33 ∷vapC−H6)or ment for an SD sequence for ectopic initiation is in agreement pRB100 (pBAD33∷yoeB) were grown exponentially in LB medium at 37 °C. At with this interpretation. time zero, 0.2% arabinose was added to induce transcription. Samples were taken before and 60′ after vapC induction. (C) Measurement of relative trans- Like other components of the translational apparatus, produc- lational initiation frequencies at elongator codons (AAA and AAG) by a dual fMet tion of tRNA is curtailed by the stringent response (31). luciferase assay. MG1655Δlon containing plasmids pQE-AAA or pQE-AAG and fMet Moreover, the charging level of tRNA decreases in response pKW3352HC (pBAD33 ∷vapC−H6) or pRB100 (pBAD33∷yoeB) were grown to leucine starvation (32). Thus, bacterial cells regulate the level exponentially in LB medium at 37 °C. Samples were taken before and 30′ after of tRNAfMet in response to environmental changes. The pro- induction of vapC. Cell samples treated with chloramphenicol (50 μg∕mL) posed regulation makes physiological sense, because a reduced were included as additional controls. F/R: Firefly/Renilla activity ratio. rate of translation reduces both nutrient consumption and the translational error rate (33) that, in turn, may increase cellular of Neisseria gonorrhoaea, which reduces intracellular trafficing fitness. We showed here that VapC can be activated by abrupt during pathogenesis, thus allowing the bacteria to better evade reduction of the growth rate (Fig. 3D). Thus vapBC may reduce the immune system (35, 36). The work presented here has opened the translational error rate during conditions of slow growth sim- the path to identify the cellular targets of VapC PIN-domain ply by reducing the drain on charged tRNA. In addition, it cannot be excluded that vapBC induces major changes in the proteome proteins from other organisms, including M. tuberculosis. of slowly growing or starved cells, although this remains to Materials and Methods be seen. Bacterial strains were grown in LB medium at 37 °C. When appropriate, the We propose that enteric VapCs represent a unique class of growth medium was supplemented with 30 μg∕mL or 100 μg∕mL of ampicil- tRNases that are beneficial during environmental stresses. We lin for low and high-copy-number plasmids, respectively or 50 μg∕mL of do not exclude that vapBC TA loci play additional roles in chloramphenicol. Transcription from LacI-regulated promoters was induced enterics or in other organisms. However, a function as stress by the addition of 2 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG). response elements is supported by studies showing that stressful Transcription from AraC-regulated promoters was induced by the addition conditions activated vapBC transcription in M. tuberculosis and of 0.2% arabinose. Strains and plasmids used and constructed are listed in S. solfataricus (11, 34). In one case, a vapBC mutant strain Table S1 and oligonucleotides in Table S2. For a full description of Materials became temperature sensitive (34). A further example is fitAB and Methods,seeSI Text

7406 ∣ www.pnas.org/cgi/doi/10.1073/pnas.1019587108 Winther and Gerdes Downloaded by guest on September 26, 2021 ACKNOWLEDGMENTS. We thank Ditlev Brodersen & Michael Sørensen for for the gift of charged formyl-methionine tRNA. This work was supported critical reading of the manuscript, Daniel Castro-Roa and Nikolay Zenkin by the Wellcome Trust.

1. Fozo EM, Hemm MR, Storz G (2008) Small toxic proteins and the antisense RNAs that 20. Robson J, McKenzie JL, Cursons R, Cook GM, Arcus VL (2009) The vapBC operon from repress them. Microbiol Mol Biol Rev 72:579–589 Table. Mycobacterium smegmatis is an autoregulated toxin-antitoxin module that controls 2. Gerdes K, Wagner EG (2007) RNA antitoxins. Curr Opin Microbiol 10:117–124. growth via inhibition of translation. J Mol Biol 390:353–367. 3. Gerdes K, Christensen SK, Lobner-Olesen A (2005) Prokaryotic toxin-antitoxin stress 21. Winther KS, Gerdes K (2009) Ectopic production of VapCs from Enterobacteria inhibits response loci. Nat Rev Microbiol 3:371–382. translation and trans-activates YoeB mRNA interferase. Mol Microbiol 72:918–930. 4. Fineran PC, et al. (2009) The phage abortive infection system, ToxIN, functions as a 22. Daines DA, Wu MH, Yuan SY (2007) VapC-1 of nontypeable Haemophilus influenzae is protein-RNA toxin-antitoxin pair. Proc Natl Acad Sci USA 106:894–899. a ribonuclease. J Bacteriol 189:5041–5048. 5. Pandey DP, Gerdes K (2005) Toxin—antitoxin loci are highly abundant in free-living but 23. Radnedge L, Davis MA, Youngren B, Austin SJ (1997) Plasmid maintenance functions of lost from host-associated prokaryotes. Nucleic Acids Res 33:966–976. the large virulence plasmid of Shigella flexneri. J Bacteriol 179:3670–3675. 6. Neubauer C, et al. (2009) The structural basis for mRNA recognition and cleavage by 24. Kaufmann G (2000) Anticodon nucleases. Trends Biochem Sci 25:70–74. the ribosome-dependent endonuclease RelE. Cell 139:1084–1095. 25. Seong BL, RajBhandary UL (1987) Escherichia coli formylmethionine tRNA: mutations 7. Pedersen K, et al. (2003) The bacterial toxin RelE displays codon-specific cleavage of in GGGCCC sequence conserved in anticodon stem of initiator tRNAs affect initiation mRNAs in the ribosomal A site. Cell 112:131–140. of protein synthesis and conformation of anticodon loop. Proc Natl Acad Sci USA 8. Christensen SK, Gerdes K (2003) RelE toxins from bacteria and Archaea cleave mRNAs 84:334–338. on translating ribosomes, which are rescued by tmRNA. Mol Microbiol 48:1389–1400. 26. Christensen-Dalsgaard M, Gerdes K (2008) Translation affects YoeB and MazF 9. Andreev D, et al. (2008) The bacterial toxin RelE induces specific mRNA cleavage in the messenger RNA interferase activities by different mechanisms. Nucleic Acids Res A site of the eukaryote ribosome. RNA 14:233–239. 36:6472–6481. 10. Temperley R, Richter R, Dennerlein S, Lightowlers RN, Chrzanowska-Lightowlers ZM 27. Tomita K, Ogawa T, Uozumi T, Watanabe K, Masaki H (2000) A cytotoxic ribonuclease (2010) Hungry codons promote frameshifting in human mitochondrial ribosomes. which specifically cleaves four isoaccepting arginine tRNAs at their anticodon loops. Science 327:301. Proc Natl Acad Sci USA 97:8278–8283. 11. Ramage HR, Connolly LE, Cox JS (2009) Comprehensive functional analysis of 28. Ogawa T, et al. (1999) A cytotoxic ribonuclease targeting specific transfer RNA Mycobacterium tuberculosis toxin-antitoxin systems: implications for pathogenesis, anticodons. Science 283:2097–2100. stress responses, and evolution. PLoS Genet 5:e1000767. 29. Amitsur M, Levitz R, Kaufmann G (1987) Bacteriophage T4 anticodon nuclease, 12. Jørgensen MG, Pandey DP, Jaskolska M, Gerdes K (2009) HicA of Escherichia coli polynucleotide kinase and RNA ligase reprocess the host lysine tRNA. EMBO J defines a novel family of translation-independent mRNA interferases in bacteria 6:2499–2503. and archaea. J Bacteriol 191:1191–1199. 30. Kapoor S, Das G, Varshney U (2011) Crucial contribution of the multiple copies of the 13. Arcus VL, Backbro K, Roos A, Daniel EL, Baker EN (2004) Distant structural homology initiator tRNA genes in the fidelity of tRNA(fMet) selection on the ribosomal P-site in leads to the functional characterization of an archaeal PIN domain as an exonuclease. Escherichia coli. Nucleic Acids Res 39:202–212. J Biol Chem 279:16471–16478. 31. Nagase T, Ishii S, Imamoto F (1988) Differential transcriptional control of the two tRNA 14. Clissold PM, Ponting CP (2000) PIN domains in nonsense-mediated mRNA decay and (fMet) genes of Escherichia coli K-12. Gene 67:49–57. RNAi. Curr Biol 10:R888–R890. 32. Dittmar KA, Sørensen MA, Elf J, Ehrenberg M, Pan T (2005) Selective charging of tRNA 15. Glavan F, Behm-Ansmant I, Izaurralde E, Conti E (2006) Structures of the PIN domains isoacceptors induced by amino-acid starvation. EMBO Rep 6:151–157. of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex. EMBO J 33. Sørensen MA, Jensen KF, Pedersen S (1994) High concentrations of ppGpp decrease the 25:5117–5125. RNA chain growth rate. Implications for protein synthesis and translational fidelity 16. Pertschy B, et al. (2009) RNA helicase Prp43 and its co-factor Pfa1 promote 20 to 18 S during amino acid starvation in Escherichia coli. J Mol Biol 236:441–454. rRNA processing catalyzed by the endonuclease Nob1. J Biol Chem 284:35079–35091. 34. Cooper CR, Daugherty AJ, Tachdjian S, Blum PH, Kelly RM (2009) Role of vapBC 17. Schneider C, Leung E, Brown J, Tollervey D (2009) The N-terminal PIN domain of the toxin-antitoxin loci in the thermal stress response of Sulfolobus solfataricus. Biochem exosome subunit Rrp44 harbors endonuclease activity and tethers Rrp44 to the yeast Soc Trans 37:123–126. core exosome. Nucleic Acids Res 37:1127–1140. 35. Mattison K, Wilbur JS, So M, Brennan RG (2006) Structure of FitAB from Neisseria 18. Schaeffer D, et al. (2009) The exosome contains domains with specific endoribonu- gonorrhoeae bound to DNA reveals a tetramer of toxin-antitoxin heterodimers clease, exoribonuclease and cytoplasmic mRNA decay activities. Nat Struct Mol Biol containing pin domains and ribbon-helix-helix motifs. J Biol Chem 281:37942–37951. 16:56–62. 36. Hopper S, et al. (2000) Isolation of Neisseria gonorrhoeae mutants that show 19. Skruzny M, et al. (2009) An endoribonuclease functionally linked to perinuclear mRNP enhanced trafficking across polarized T84 epithelial monolayers. Infect Immun quality control associates with the nuclear pore complexes. PLoS Biol 7:e8. 68:896–905. BIOCHEMISTRY

Winther and Gerdes PNAS ∣ May 3, 2011 ∣ vol. 108 ∣ no. 18 ∣ 7407 Downloaded by guest on September 26, 2021