Interaction studies on bacterial stringent response protein RelA with uncharged tRNA provide evidence for its prerequisite complex for ribosome binding

Abstract

The bacterial stringent response is regulated by the synthesis of (p)ppGpp which is mediated by RelA in a complex with uncharged tRNA and ribosome. We intended to probe RelA–uncharged tRNA interactions off the ribosome to understand the sequential activation mechanism of RelA. Stringent response is a key regulatory pleiotropic mechanism which allows bacteria to survive in unfavorable conditions. Since the discovery of RelA, it has been believed that it is activated upon binding to ribosomes which already have uncharged tRNA on acceptor site (A-site). However, uncharged tRNA occupied in the A-site of the ribosome prior to RelA binding could not be observed; therefore, recently an alternate model for RelA activation has been proposed in which RelA first binds to uncharged tRNA and then RelA–uncharged tRNA complex is loaded on to the ribosome to synthesize (p)ppGpp. To explore the alternate hypothesis, we report here the in vitro binding of uncharged tRNA to RelA in the absence of ribosome using formaldehyde cross-linking, fluorescence spectroscopy, surface plasmon resonance, size-exclusion chromatography, and hydrogen–deuterium exchange mass spectrometry. Altogether, our results clearly indicate binding between RelA and uncharged tRNA without the involvement of ribosome. Moreover, we have analyzed their binding kinetics and mapping of tRNA-interacting regions of RelA structure. We have also co-purified TGS domain in complex with tRNA to further establish in vivo RelA–tRNA binding. We have observed that TGS domain recognizes all types of uncharged tRNA similar to EF-Tu and tRNA interactions. Altogether, our results demonstrate the complex formation between RelA and uncharged tRNA that may be loaded to the ribosome for (p)ppGpp synthesis.

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References

  1. Agirrezabala X, Fernández IS, Kelley AC et al (2013) The ribosome triggers the stringent response by RelA via a highly distorted tRNA. EMBO Rep 14:811–816. https://doi.org/10.1038/embor.2013.106

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Arenz S, Abdelshahid M, Sohmen D et al (2016) The stringent factor RelA adopts an open conformation on the ribosome to stimulate ppGpp synthesis. Nucleic Acids Res 44:6471–6481. https://doi.org/10.1093/nar/gkw470

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Atkinson GC, Tenson T, Hauryliuk V (2011) The RelA/SpoT homolog (RSH) superfamily: distribution and functional evolution of ppGpp synthetases and hydrolases across the tree of life. PLoS One 6:e23479. https://doi.org/10.1371/journal.pone.0023479

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Balaban NQ, Gerdes K, Lewis K, McKinney JD (2013) A problem of persistence: still more questions than answers? Nat Rev Microbiol 11:587–591

    Article  CAS  PubMed  Google Scholar 

  5. Barker MM, Gaal T, Josaitis CA, Gourse RL (2001) Mechanism of regulation of transcription initiation by ppGpp. I. Effects of ppGpp on transcription initiation in vivo and in vitro. J Mol Biol 305:673–688. https://doi.org/10.1006/JMBI.2000.4327

    Article  CAS  PubMed  Google Scholar 

  6. Berghoff BA, Wagner EGH (2017) RNA-based regulation in type I toxin-antitoxin systems and its implication for bacterial persistence. Curr Genet 63:1011–1016. https://doi.org/10.1007/s00294-017-0710-y

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Brown A, Fernández IS, Gordiyenko Y, Ramakrishnan V (2016) Ribosome-dependent activation of stringent control. Nature 534:277. https://doi.org/10.1038/nature17675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Cashel M, Gallant J (1969) Two compounds implicated in the function of the RC gene of Escherichia coli. Nature 221:838–841

    Article  CAS  PubMed  Google Scholar 

  9. Castro-Cerritos KV, Lopez-Torres A, Obregón-Herrera A et al (2018) LC–MS/MS proteomic analysis of starved Bacillus subtilis cells overexpressing ribonucleotide reductase (nrdEF): implications in stress-associated mutagenesis. Curr Genet 64:215–222. https://doi.org/10.1007/s00294-017-0722-7

    Article  CAS  PubMed  Google Scholar 

  10. Cover TL, Blaser MJ (2009) Helicobacter pylori in health and disease. Gastroenterology 136:1863–1873. https://doi.org/10.1053/j.gastro.2009.01.073

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Dalebroux ZD, Swanson MS (2012) ppGpp: magic beyond RNA polymerase. Nat Rev Microbiol 10:203–212. https://doi.org/10.1038/nrmicro2720

    Article  CAS  PubMed  Google Scholar 

  12. Dalebroux ZD, Svensson SL, Gaynor EC, Swanson MS (2010) ppGpp conjures bacterial virulence. Microbiol Mol Biol Rev 74:171–199. https://doi.org/10.1128/MMBR.00046-09

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Dye C, Scheele S, Dolin P et al (1999) Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO Global Surveillance and Monitoring Project. JAMA 282:677–686

    Article  CAS  Google Scholar 

  14. Eckel M, Steinchen W, Batschauer A (2018) ATP boosts lit state formation and activity of Arabidopsis cryptochrome 2. Plant J. https://doi.org/10.1111/tpj.14039

    Article  PubMed  Google Scholar 

  15. Fast R, Sköld O (1977) Biochemical mechanism of uracil uptake regulation in Escherichia coli B. Allosteric effects on uracil phosphoribosyltransferase under stringent conditions. J Biol Chem 252:7620–7624

    CAS  PubMed  Google Scholar 

  16. Geiger T, Francois P, Liebeke M et al (2012) The stringent response of staphylococcus aureus and its impact on survival after phagocytosis through the induction of intracellular PSMs expression. PLoS Pathog 8:e1003016. https://doi.org/10.1371/journal.ppat.1003016

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Geromanos SJ, Vissers JPC, Silva JC et al (2009) The detection, correlation, and comparison of peptide precursor and product ions from data independent LC-MS with data dependant LC–MS/MS Keywords: biomarker discovery/data-independent LC–MS/multiplexed LC–MS/shotgun sequencing/time-resolved mass spectrometry. Proteomics 9:1683–1695. https://doi.org/10.1002/pmic.200800562

    Article  CAS  PubMed  Google Scholar 

  18. Gohara DW, Yap M-NF (2018) Survival of the drowsiest: the hibernating 100S ribosome in bacterial stress management. Curr Genet 64:753–760. https://doi.org/10.1007/s00294-017-0796-2

    Article  CAS  Google Scholar 

  19. Gong F, Fahy D, Smerdon MJ (2006) Combination of chemical cross-linking and pull-down assay to study transient protein–protein interactions. Protoc Exch. https://doi.org/10.1038/nprot.2006.297

    Article  Google Scholar 

  20. Grant SS, Hung DT (2013) Persistent bacterial infections, antibiotic tolerance, and the oxidative stress response. Virulence 4:273–283. https://doi.org/10.4161/viru.23987

    Article  PubMed  PubMed Central  Google Scholar 

  21. Harms A, Maisonneuve E, Gerdes K (2016) Mechanisms of bacterial persistence during stress and antibiotic exposure. Science. https://doi.org/10.1126/science.aaf4268

    Article  PubMed  PubMed Central  Google Scholar 

  22. Haseltine WA, Block R (1973) Synthesis of guanosine tetra- and pentaphosphate requires the presence of a codon-specific, uncharged transfer ribonucleic acid in the acceptor site of ribosomes. Proc Natl Acad Sci USA 70:1564–1568

    Article  CAS  PubMed  Google Scholar 

  23. Hauenstein SI, Hou Y-M, Perona JJ (2008) The homotetrameric phosphoseryl-tRNA synthetase from Methanosarcina mazei exhibits half-of-the-sites activity. J Biol Chem 283:21997–22006. https://doi.org/10.1074/jbc.M801838200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Hauryliuk V, Atkinson GC, Murakami KS et al (2015) Recent functional insights into the role of (p)ppGpp in bacterial physiology. Nat Rev Microbiol 13:298–309. https://doi.org/10.1038/nrmicro3448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Hogg T, Mechold U, Malke H et al (2004) Conformational antagonism between opposing active sites in a bifunctional RelA/SpoT homolog modulates (p)ppGpp metabolism during the stringent response [corrected]. Cell 117:57–68. https://doi.org/10.1016/S0092-8674(04)00260-0

    Article  CAS  PubMed  Google Scholar 

  26. Kanjee U, Gutsche I, Alexopoulos E et al (2011) Linkage between the bacterial acid stress and stringent responses: the structure of the inducible lysine decarboxylase. EMBO J 30:931–944. https://doi.org/10.1038/emboj.2011.5

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Kanjee U, Ogata K, Houry WA (2012) Direct binding targets of the stringent response alarmone (p)ppGpp. Mol Microbiol 85:1029–1043. https://doi.org/10.1111/j.1365-2958.2012.08177.x

    Article  CAS  PubMed  Google Scholar 

  28. Knutsson Jenvert R-M, Holmberg Schiavone L (2005) Characterization of the tRNA and ribosome-dependent pppGpp-synthesis by recombinant stringent factor from Escherichia coli. FEBS J 272:685–695. https://doi.org/10.1111/j.1742-4658.2004.04502.x

    Article  CAS  PubMed  Google Scholar 

  29. Kriel A, Bittner AN, Kim SH et al (2012) Direct regulation of GTP homeostasis by (p)ppGpp: a critical component of viability and stress resistance. Mol Cell 48:231–241. https://doi.org/10.1016/j.molcel.2012.08.009

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Kudrin P, Varik V, Oliveira SRA et al (2017) Subinhibitory concentrations of bacteriostatic antibiotics induce relA—dependent and relA—independent tolerance to β-lactams. Antimicrob Agents Chemother 61:e02173-16. https://doi.org/10.1128/AAC.02173-16

    Article  PubMed  PubMed Central  Google Scholar 

  31. Kudrin P, Dzhygyr I, Ishiguro K et al (2018) The ribosomal A-site finger is crucial for binding and activation of the stringent factor RelA. Nucleic Acids Res 46:1973–1983. https://doi.org/10.1093/nar/gky023

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kussell E, Kishony R, Balaban NQ, Leibler S (2005) Bacterial persistence: a model of survival in changing environments. Genetics 169:1807–1814. https://doi.org/10.1534/genetics.104.035352

    Article  PubMed  PubMed Central  Google Scholar 

  33. Legault L, Jeantet C, Gros F (1972) Inhibition of in vitro protein synthesis by ppGpp. FEBS Lett 27:71–75. https://doi.org/10.1016/0014-5793(72)80412-5

    Article  CAS  PubMed  Google Scholar 

  34. Li G-Z, Vissers JPC, Silva JC et al (2009) Database searching and accounting of multiplexed precursor and product ion spectra from the data independent analysis of simple and complex peptide mixtures. Proteomics 9:1696–1719. https://doi.org/10.1002/pmic.200800564

    Article  CAS  PubMed  Google Scholar 

  35. Louie A, Jurnak F (1985) Kinetic studies of Escherichia coli elongation factor Tu-guanosine 5′-triphosphate-aminoacyl-tRNA complexes. Biochemistry 24:6433–6439. https://doi.org/10.1021/bi00344a019

    Article  CAS  PubMed  Google Scholar 

  36. Loveland AB, Bah E, Madireddy R et al (2016) Ribosome·RelA structures reveal the mechanism of stringent response activation. Elife. https://doi.org/10.7554/elife.17029

    Article  PubMed  PubMed Central  Google Scholar 

  37. Maisonneuve E, Gerdes K (2014) Molecular mechanisms underlying bacterial persisters. Cell 157:539–548. https://doi.org/10.1016/j.cell.2014.02.050

    Article  CAS  PubMed  Google Scholar 

  38. Masek T, Vopalensky V, Suchomelova P, Pospisek M (2005) Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem 336:46–50. https://doi.org/10.1016/J.AB.2004.09.010

    Article  CAS  PubMed  Google Scholar 

  39. Mechold U, Potrykus K, Murphy H et al (2013) Differential regulation by ppGpp versus pppGpp in Escherichia coli. Nucleic Acids Res 41:6175–6189. https://doi.org/10.1093/nar/gkt302

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Milon P, Tischenko E, Tomsic J et al (2006) The nucleotide-binding site of bacterial translation initiation factor 2 (IF2) as a metabolic sensor. Proc Natl Acad Sci 103:13962–13967. https://doi.org/10.1073/pnas.0606384103

    Article  CAS  PubMed  Google Scholar 

  41. Murphy H, Cashel M (2003) Isolation of RNA polymerase suppressors of a (p)ppGpp deficiency. Methods Enzymol 371:596–601. https://doi.org/10.1016/S0076-6879(03)71044-1

    Article  CAS  PubMed  Google Scholar 

  42. Nazir A, Harinarayanan R (2015) Inactivation of cell division protein FtsZ by SulA makes lon indispensable for the viability of a ppGpp0 strain of Escherichia coli. J Bacteriol 198:688–700. https://doi.org/10.1128/JB.00693-15

    Article  CAS  PubMed  Google Scholar 

  43. Nazir A, Harinarayanan R (2016) (p)ppGpp and the bacterial cell cycle. J Biosci 41:277–282

    Article  CAS  PubMed  Google Scholar 

  44. Pao CC, Dyes BT (1981) Effect of unusual guanosine nucleotides on the activities of some Escherichia coli cellular enzymes. Biochim Biophys Acta Gen Subj 677:358–362. https://doi.org/10.1016/0304-4165(81)90247-6

    Article  CAS  Google Scholar 

  45. Payoe R, Fahlman RP (2011) Dependence of RelA-mediated (p)ppGpp formation on tRNA identity. Biochemistry 50:3075–3083. https://doi.org/10.1021/bi1015309

    Article  CAS  PubMed  Google Scholar 

  46. Persky NS, Ferullo DJ, Cooper DL et al (2009) The ObgE/CgtA GTPase influences the stringent response to amino acid starvation in Escherichia coli. Mol Microbiol 73:253–266. https://doi.org/10.1111/j.1365-2958.2009.06767.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Pollard TD (2010) A guide to simple and informative binding assays. Mol Biol Cell 21:4061–4067. https://doi.org/10.1091/mbc.E10-08-0683

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Poole K (2012) Bacterial stress responses as determinants of antimicrobial resistance. J Antimicrob Chemother 67:2069–2089. https://doi.org/10.1093/jac/dks196

    Article  CAS  PubMed  Google Scholar 

  49. Potrykus K, Cashel M (2008) (p)ppGpp: still magical? Annu Rev Microbiol 62:35–51. https://doi.org/10.1146/annurev.micro.62.081307.162903

    Article  CAS  PubMed  Google Scholar 

  50. Prusa J, Zhu DX, Stallings CL (2018) The stringent response and Mycobacterium tuberculosis pathogenesis. Pathog Dis. https://doi.org/10.1093/femspd/fty054

    Article  PubMed  Google Scholar 

  51. Ramagopal S, Davis BD (1974) Localization of the stringent protein of Escherichia coli on the 50S ribosomal subunit. Proc Natl Acad Sci USA 71:820–824

    Article  CAS  PubMed  Google Scholar 

  52. Rhen M, Eriksson S, Clements M et al (2003) The basis of persistent bacterial infections. Trends Microbiol 11:80–86. https://doi.org/10.1016/S0966-842X(02)00038-0

    Article  CAS  PubMed  Google Scholar 

  53. Richter D (1976) Stringent factor from Escherichia coli directs ribosomal binding and release of uncharged tRNA. Proc Natl Acad Sci USA 73:707–711

    Article  CAS  PubMed  Google Scholar 

  54. Richter D, Nowak P, Kleinert U (1975) Escherichia coli stringent factor binds to ribosomes at a site different from that of elongation factor Tu or G. Biochemistry 14:4414–4420. https://doi.org/10.1021/bi00691a012

    Article  CAS  PubMed  Google Scholar 

  55. Rojas AM, Ehrenberg M, Andersson SG, Kurland CG (1984) ppGpp inhibition of elongation factors Tu, G and Ts during polypeptide synthesis. Mol Gen Genet 197:36–45

    Article  CAS  PubMed  Google Scholar 

  56. Ross W, Vrentas CE, Sanchez-Vazquez P et al (2013) The magic spot: a ppGpp binding site on E. coli RNA polymerase responsible for regulation of transcription initiation. Mol Cell 50:420–429. https://doi.org/10.1016/J.MOLCEL.2013.03.021

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Schäper S, Steinchen W, Krol E et al (2017) AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production. Proc Natl Acad Sci 114:E4822–E4831. https://doi.org/10.1073/pnas.1702435114

    Article  CAS  PubMed  Google Scholar 

  58. Srivatsan A, Wang JD (2008) Control of bacterial transcription, translation and replication by (p)ppGpp. Curr Opin Microbiol 11:100–105. https://doi.org/10.1016/j.mib.2008.02.001

    Article  CAS  PubMed  Google Scholar 

  59. Steinchen W, Bange G (2016) The magic dance of the alarmones (p)ppGpp. Mol Microbiol 101:531–544. https://doi.org/10.1111/mmi.13412

    Article  CAS  PubMed  Google Scholar 

  60. Steinchen W, Schuhmacher JS, Altegoer F et al (2015) Catalytic mechanism and allosteric regulation of an oligomeric (p) ppGpp synthetase by an alarmone. Proc Natl Acad Sci 112:1–6. https://doi.org/10.1073/pnas.1505271112

    Article  CAS  Google Scholar 

  61. Wales TE, Fadgen KE, Gerhardt GC, Engen JR (2008) High-speed and high-resolution UPLC separation at zero degrees celsius. Anal Chem 80:6815–6820. https://doi.org/10.1021/ac8008862

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Walker SE, Fredrick K (2008) Preparation and evaluation of acylated tRNAs. Methods 44:81–86. https://doi.org/10.1016/j.ymeth.2007.09.003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Wang JD, Sanders GM, Grossman AD (2007) Nutritional control of elongation of DNA replication by (p)ppGpp. Cell 128:865–875. https://doi.org/10.1016/j.cell.2006.12.043

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Wendrich TM, Blaha G, Wilson DN et al (2002) Dissection of the mechanism for the stringent factor RelA. Mol Cell 10:779–788. https://doi.org/10.1016/S1097-2765(02)00656-1

    Article  CAS  PubMed  Google Scholar 

  65. Winther KS, Roghanian M, Gerdes K (2018) Activation of the stringent response by loading of RelA–tRNA complexes at the ribosomal A-Site. Mol Cell 70:95–105.e4. https://doi.org/10.1016/j.molcel.2018.02.033

    Article  CAS  PubMed  Google Scholar 

  66. Young D, Hussell T, Dougan G (2002) Chronic bacterial infections: living with unwanted guests. Nat Immunol 3:1026–1032. https://doi.org/10.1038/ni1102-1026

    Article  CAS  PubMed  Google Scholar 

  67. Zhang C-M, Perona JJ, Ryu K et al (2006) Distinct kinetic mechanisms of the two classes of aminoacyl-tRNA synthetases. J Mol Biol 361:300–311. https://doi.org/10.1016/J.JMB.2006.06.015

    Article  CAS  PubMed  Google Scholar 

  68. Zhang Y, Zborníková E, Rejman D, Gerdes K (2018) Novel (p)ppGpp binding and metabolizing proteins of Escherichia coli. MBio. https://doi.org/10.1128/mbio.02188-17

    Article  PubMed  PubMed Central  Google Scholar 

  69. Zuo Y, Wang Y, Steitz TA (2013) The mechanism of E. coli RNA polymerase regulation by ppGpp is suggested by the structure of their complex. Mol Cell 50:430–436. https://doi.org/10.1016/J.MOLCEL.2013.03.020

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work is funded by Science and Engineering Research Board (SERB), Government of India under Young Scientist Start-up Grant (SB/YS/LS-176/2014) and also supported by EMBO Short-term fellowship to GSK. We thank Mr. Manu Vashist and Advance Instrumentation Research Facility, Jawaharlal Nehru University (JNU), for providing the SPR facility. We thank Wieland Steinchen from the HDX-MS core facility MIDAS (Marburg) for performing the HDX-MS experiments. We thank ICGEB core funds.

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Correspondence to Gajraj Singh Kushwaha or Neel Sarovar Bhavesh.

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Kushwaha, G.S., Bange, G. & Bhavesh, N.S. Interaction studies on bacterial stringent response protein RelA with uncharged tRNA provide evidence for its prerequisite complex for ribosome binding. Curr Genet 65, 1173–1184 (2019). https://doi.org/10.1007/s00294-019-00966-y

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Keywords

  • Stringent response
  • RelA
  • ppGpp synthetase
  • tRNA–protein interaction