RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis

•Several families of toxSAS RSH toxins specifically inhibit protein synthesis•Translation-inhibiting toxSASs transfer pyrophosphate from ATP to the tRNA 3′ CCA•3′ CCA pyrophosphorylation abrogates aminoacylation•Some SAH enzymes can reverse RelA-SpoT Homolog (RSH) control bacterial physiology through synthesis and degradation nucleotide alarmone (p)ppGpp. We recently discovered multiple small synthetase (SAS) acting as toxin-antitoxin (TA) modules, with FaRel subfamily abrogating growth by producing an analog (p)ppGpp, (pp)pApp. Here we probe mechanism arrest used four experimentally unexplored subfamilies toxSAS: FaRel2, PhRel, PhRel2, CapRel. Surprisingly, all these synthesis. To do so, they a moiety CCA. The modification inhibits both aminoacylation sensing cellular amino acid starvation ribosome-associated RelA. Conversely, show that some hydrolase (SAH) counter inhibition toxSAS. Collectively, establish RSHs RNA-modifying enzymes. Toxin-antitoxin systems are class highly diverse widespread operons found in bacterial, archaeal, bacteriophage genomes (Fraikin et al., 2020Fraikin N. Goormaghtigh F. Van Melderen L. Type II systems: evolution revolutions.J. Bacteriol. 2020; 202 (e00763-19)Crossref PubMed Google Scholar; Harms 2018Harms A. Brodersen D.E. Mitarai Gerdes K. Toxins, targets, triggers: overview biology.Mol. Cell. 2018; 70: 768-784Abstract Full Text PDF Scopus (266) Scholar). TAs have broad range functions, including defense against bacteriophages, phage competition for infection bacteria, stabilization transposons, plasmids, genomes, which rely on potent toxicity toxin controlled protein- or RNA-based antitoxin (Blower 2009Blower T.R. Fineran P.C. Johnson M.J. Toth I.K. Humphreys D.P. Salmond G.P. Mutagenesis functional characterization RNA components toxIN abortive locus Erwinia.J. 2009; 191: 6029-6039Crossref (56) Fiedoruk 2015Fiedoruk Daniluk T. Swiecicka I. Sciepuk M. Leszczynska unevenly distributed among Escherichia coli phylogroups.Microbiology (Reading). 2015; 161: 158-167Crossref (30) Guegler Laub, 2021Guegler C.K. Laub M.T. Shutoff host transcription triggers system cleave abort infection.Mol. 2021; 81: 2361-2373.e9Abstract (8) Jaffé 1985Jaffé Ogura Hiraga S. Effects ccd function F plasmid growth.J. 1985; 163: 841-849Crossref Lima-Mendez 2020Lima-Mendez G. Oliveira Alvarenga D. Ross Hallet B. Varani A.M. Chandler gene pairs Tn 3 family transposons appear be integral part transposition module.MBio. 11 (e00452-20)Crossref (12) Song Wood, 2020Song Wood T.K. A primary physiological role toxin/antitoxin is inhibition.Front. Microbiol. 11: 1895Crossref (21) Many TA closely evolutionary related housekeeping enzymes, suggesting “breakaway” path generally harmless enzyme evolves toxic requires tight (Burckhardt Escalante-Semerena, 2020Burckhardt R.M. Escalante-Semerena J.C. Small-molecule acetylation GCN5-related N-acetyltransferases bacteria.Microbiol. Mol. Biol. Rev. 84 (e00090-19)Crossref (10) Garcia-Pino 2014Garcia-Pino Zenkin Loris R. many faces Fic: structural aspects Fic enzymes.Trends Biochem. Sci. 2014; 39: 121-129Abstract (48) Jimmy 2020Jimmy Saha Kurata Stavropoulos C. S.R.A. Koh Cepauskas Takada H. Rejman Tenson al.A exploiting via signaling.Proc. Natl. Acad. U S 117: 10500-10510Crossref (28) Koonin Makarova, 2019Koonin E.V. Makarova K.S. Origins CRISPR-Cas systems.Philos. Trans. Soc. Lond. B 2019; 374: 20180087Crossref (92) Senissar 2017Senissar Manav M.C. Structural conservation PIN domain active site across domains life.Protein 2017; 26: 1474-1492Crossref (18) stress-response was only recognized also contain (Jimmy levels nucleotides ppGpp (guanosine-3ʹ,5ʹ-bisdiphosphate) pppGpp (guanosine-5ʹ-triphosphate-3ʹ-diphosphate), collectively referred alarmones, turn, regulating metabolism, virulence, rate, well playing important antibiotic stress tolerance (Gaca 2015Gaca A.O. Colomer-Winter Lemos J.A. means common end: intricacies (p)ppGpp metabolism its homeostasis.J. 197: 1146-1156Crossref (123) Hauryliuk 2015Hauryliuk V. Atkinson G.C. Murakami Recent insights into physiology.Nat. 13: 298-309Crossref (410) Irving 2021Irving S.E. Choudhury N.R. Corrigan stringent response roles (pp)pGpp bacteria.Nat. 19: 256-271Crossref (29) Liu 2015Liu Bittner A.N. Wang J.D. Diversity effectors.Curr. Opin. 24: 72-79Crossref (114) Zhu 2019Zhu Pan Y. Dai X. (p)ppGpp: magic governor economy.Curr. Genet. 65: 1121-1125Crossref members synthesize transferring group 3ʹ position either GDP GTP convert it back GDP/GTP removal (Atkinson 2011Atkinson RelA/SpoT homolog superfamily: distribution synthetases hydrolases tree life.PLoS ONE. 2011; 6: e23479Crossref (253) Cashel Gallant, 1969Cashel Gallant J. Two compounds implicated RC coli.Nature. 1969; 221: 838-841Crossref (454) classified long multi-domain RSHs, archetypical representatives being RelA (Haseltine Block, 1973Haseltine W.A. Block Synthesis guanosine tetra- pentaphosphate presence codon-specific, uncharged ribonucleic acceptor ribosomes.Proc. 1973; 1564-1568Crossref (332) Scholar) SpoT (Xiao 1991Xiao Kalman Ikehara Zemel Glaser Residual 3′,5′-bispyrophosphate synthetic activity relA null mutants eliminated spoT mutations.J. Chem. 1991; 266: 5980-5990Abstract Scholar), short single-domain latter subdivided (SASs; 30 distinct subfamilies, several characterized experimentally, Staphylococcus aureus RelP [Geiger 2014Geiger Kästle Gratani F.L. Goerke Wolz synthases mediate cell envelope conditions.J. 196: 894-902Crossref (107) 2018Manav Beljantseva Bojer M.S. Ingmer basis RelP.J. 293: 3254-3264Abstract Scholar] Bacillus subtilis RelQ [Nanamiya 2008Nanamiya Kasai Nozawa Yun C.S. Narisawa Natori Kawamura Tozawa Identification analysis novel genes subtilis.Mol. 2008; 67: 291-304Crossref (158) Steinchen 2015Steinchen W. Schuhmacher J.S. Altegoer Fage C.D. Srinivasan Linne U. Marahiel M.A. Bange Catalytic allosteric regulation oligomeric alarmone.Proc. 112: 13348-13353Crossref (67) Scholar]) (SAHs; subfamilies) Several recent discoveries sparked interest how non-(p)ppGpp RSH-mediated chemical catalysis weaponized bacteria inhibition. Highly SAS injected secretion (Ahmad 2019Ahmad Walker M.D. Tran H.R. Stogios P.J. Savchenko Grant R.A. McArthur A.G. Whitney An interbacterial target synthesizing (p)ppApp.Nature. 575: 674-678Crossref (42) constitute (toxSAS) modules were produce (p)ppGpp—pApp, ppApp, pppApp—collectively (pp)pApp (Figure S1). By de novo purine orthosteric PurF translation, transcription, replication SAHs been shown catalyze unexpected non-alarmone reactions; while no encoded mammalian human MESH1 identified 2010 efficient (Sun 2010Sun Lee J.H. Kim H.Y. Rhee H.W. Park S.Y. K.J. B.Y. Hong J.I. metazoan ortholog hydrolyzes functions responses.Nat. Struct. 2010; 17: 1188-1194Crossref (66) decade later, compelling evidence presented NADPH phosphatase (Ding 2020Ding C.C. Rose Sun Wu Chen P.H. Lin Yang W.H. K.Y. Xu E. al.MESH1 cytosolic regulates ferroptosis.Nat. Metab. 2: 270-277Crossref (36) Combined dramatic diversity this largely demonstrate versatile biological clearly not limited metabolism. validated five bona fide effectors: Cellulomonas marina FaRel, la1a Coprobacillus sp. D7 Mycobacterium Phrann PhRel (Gp29), tuberculosis AB308 CapRel Out these, (pp)pApp-producing previously functionally characterized. In study uncover surprising reaction catalyzed shed light counteracted hydrolytic SAHs. Although yet uncharacterized, initially assumed toxSASs, just Tas1 effector Pseudomonas aeruginosa VI when analyzed pools growth-arrested expressing detected accumulation 1A). At same time, robustly upon expression S1E; standards described Figure S2). Similarly, did detect FaRel2 S1H). These results suggested might universally act production (pp)pApp, therefore, could Tas1. metabolic labeling effects uncharacterized translation (by following incorporation 35S-methionine proteins), (incorporation 3H-uridine RNA), 3H-thymidine DNA). stark contrast three processes S3A), inhibited translation. strongest observed PhRel2 (Figures 1B 1C), superinfection immunity (Gp29) (Dedrick 2017Dedrick Jacobs-Sera Bustamante C.A. Garlena Mavrich T.N. Pope Reyes Russell D.A. Adair Alvey al.Prophage-mediated defence viral attack counter-defence.Nat. 16251Crossref (94) had weaker but still specific effect S3B S3C). Interestingly, induction increased. This likely due abrogation consumption cessation resulting increased transcription; earlier similar kanamycin Furthermore, pronounced polysomal fraction S3D), elongation stalled antibiotics (Ennis, 1972Ennis H.L. Polysome coli: polysome stability.Antimicrob. Agents Chemother. 1972; 1: 197-203Crossref (15) our mode toxicity, deviating modus operandi. next tested whether mediated direct using dihydrofolate reductase (DHFR) reconstituted cell-free (PURE) (Shimizu 2001Shimizu Inoue Tomari Suzuki Yokogawa Nishikawa Ueda Cell-free purified components.Nat. Biotechnol. 2001; 751-755Crossref (1214) readout activity. purification exceedingly challenging succeeded purifying enzymatically competent C-terminally FLAG3-tagged α-FLAG3 immunoprecipitation S4). As earlier, FLAG3 tag does interfere ability counteract specificity control, catalytically compromised variants Y128A (predicted disrupt stacking interaction substrate; 2018Steinchen Vogt Giammarinaro P.I. Horvatek P. mechanistic divergence RelQ.Sci. Rep. 8: 2195Crossref (27) D90G compromise coordination Mg2+ ion; Both substituted residues conserved S5A), mutant non-toxic expressed 1D). addition wild-type, Y128A, PURE abrogated DHFR 1E; S6A). ATfaRel2 antitoxin, acts though sequestering inactive complex inhibitory although fully 1F). concluded indeed directly targets machinery. Inhibition systems, often tRNA, such cleavage (used VapC toxins; Cruz 2015Cruz J.W. Sharp Hoffer E.D. Maehigashi Vvedenskaya I.O. Konkimalla Husson R.N. Nickels B.E. Dunham C.M. Woychik N.A. Growth-regulating VapC-mt4 isoacceptor-specific tRNase.Nat. Commun. 7480Crossref (54) Winther Gerdes, 2011Winther Enteric virulence associated initiator tRNA.Proc. 108: 7403-7407Crossref (188) attached (as seen GNAT Cheverton 2016Cheverton Gollan Przydacz Wong C.T. Mylona Hare S.A. Helaine salmonella promotes persister formation tRNA.Mol. 2016; 63: 86-96Abstract (133) Jurėnas 2017Jurėnas Chatterjee Konijnenberg Sobott Droogmans AtaT blocks initiation N-acetylation tRNAfMet.Nat. 640-646Crossref (49) inactivation end pyrimidines MenT3; Cai 2020Cai Usher Gutierrez Tolcan Mansour Condon Neyrolles O. Genevaux Blower nucleotidyltransferase stems.Sci. Adv. eabb6651Crossref never any other products than hyperphosphorylated However, one imagine donor transferred onto ribose terminal adenosine instead corresponding ATP/ADP substrate Tas1/FaRel availability hydroxyl essential (Weinger Strobel, 2006Weinger Strobel Participation A76 groups throughout translation.Biochemistry. 2006; 45: 5939-5948Crossref (46) would efficiently hypothesis deacylated γ-32P radioactively labeled moiety. ATP, radiolabels tRNAifMet 2A 2B ) elongator tRNAPhe 2B). test generality toxSAS-mediated modification, PhRel2. Similarly 2C), γ-32P-labels 2D). nature installed capillary liquid chromatography (LC)/nano electrospray ionization mass spectrometry (ESI-MS) FaRel2-treated digested RNase T1 2E 2F; S7). Analysis untreated RNAPhe fragments assign T1-digested fragments: internal 5′ (5′OH) phosphate (3′P) groups, whereas CACCA-OH fragment has give unique molecular 2E; Figures S7A S7C). further probed higher energy collisional dissociation (HCD), sequence unequivocally confirmed assignment product ions S7D S7G). sample, abundance dramatically decreased (to 4%, relative minus sample), bearing (CACCA-PP, 74%) cyclic (CACCA>P, 22%) S7B HCD analyses occur at terminus CACCA S7E–S7G). speculate derivative produced pyrophosphorylated nucleophilic 2′OH pyrophosphate, release phosphate. analogous ppGpp, GDP-2′:3′-cyclic monophosphate, crystal structure N-terminal region Rel Streptococcus dysgalactiae subsp. equisimilis (Hogg 2004Hogg Mechold Malke Hilgenfeld Conformational antagonism between opposing sites bifunctional modulates during response.Cell. 2004; 57-68Abstract (227) tRNA-modifying lost 3A) AtFaRel2 type 3B 3C). result consistent pyrophosphorylating adenine residue acceptor. tRNAPhe. readily 3D) strictly ATP-dependent manner 3E), thus explaining translational principle, FaRel2-modified pyrophospho-tRNA (tRNA-PP) incompetent aminoacylation, actively because of, instance, stable binding ribosomal A-site factor EF-Tu. hypothesis, first titrated total near-saturating concentration (50 μM) which, reasoned, most sensitive S6B). added concentrations up 14 μM, tRNAPhe-PP S6C), alt