1887

Abstract

Small regulatory RNAs (sRNAs) are the most prominent post-transcriptional regulators in all kingdoms of life. A few of them, e.g. SR1 from , are dual-function sRNAs. SR1 acts as a base-pairing sRNA in arginine catabolism and as an mRNA encoding the small peptide SR1P in RNA degradation. Both functions of SR1 are highly conserved among 23 species of . Here, we investigate the interaction between SR1P and GapA by a combination of and methods. prediction of the structure of SR1P yielded five models, one of which was consistent with experimental circular dichroism spectroscopy data of a purified, synthetic peptide. Based on this model structure and a comparison between the 23 SR1P homologues, a series of SR1P mutants was constructed and analysed by Northern blotting and co-elution experiments. The known crystal structure of GapA was used to model SR1P onto this structure. The hypothetical SR1P binding pocket, composed of two α-helices at both termini of GapA, was investigated by constructing and assaying a number of GapA mutants in the presence and absence of wild-type or mutated SR1P. Almost all residues of SR1P located in the two highly conserved motifs are implicated in the interaction with GapA. A critical lysine residue (K332) in the C-terminal α-helix 14 of GapA corroborated the predicted binding pocket.

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2017-08-01
2020-03-28
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References

  1. Brantl S. Bacterial chromosome-encoded small regulatory RNAs. Future Microbiol 2009;4:85–103 [CrossRef][PubMed]
    [Google Scholar]
  2. Brantl S. Regulatory mechanisms employed by cis-encoded antisense RNAs. Curr Opin Microbiol 2007;10:102–109 [CrossRef][PubMed]
    [Google Scholar]
  3. Brantl S. Acting antisense: plasmid- and chromosome-encoded sRNAs from Gram-positive bacteria. Future Microbiol 2012;7:853–871 [CrossRef][PubMed]
    [Google Scholar]
  4. Brantl S, Brückner R. Small regulatory RNAs from low-GC Gram-positive bacteria. RNA Biol 2014;11:443–456 [CrossRef][PubMed]
    [Google Scholar]
  5. Gimpel M, Brantl S. Dual-function small regulatory RNAs in bacteria. Mol Microbiol 2017;103:387–397 [CrossRef][PubMed]
    [Google Scholar]
  6. Morfeldt E, Taylor D, von Gabain A, Arvidson S. Activation of alpha-toxin translation in Staphylococcus aureus by the trans-encoded antisense RNA, RNAIII. EMBO J 1995;14:4569–4577[PubMed]
    [Google Scholar]
  7. Mangold M, Siller M, Roppenser B, Vlaminckx BJ, Penfound TA et al. Synthesis of group A streptococcal virulence factors is controlled by a regulatory RNA molecule. Mol Microbiol 2004;53:1515–1527 [CrossRef][PubMed]
    [Google Scholar]
  8. Wadler CS, Vanderpool CK. A dual function for a bacterial small RNA: SgrS performs base pairing-dependent regulation and encodes a functional polypeptide. Proc Natl Acad Sci USA 2007;104:20454–20459 [CrossRef][PubMed]
    [Google Scholar]
  9. Kaito C, Saito Y, Ikuo M, Omae Y, Mao H et al. Mobile genetic element SCCmec-encoded psm-mec RNA suppresses translation of agrA and attenuates MRSA virulence. PLoS Pathog 2013;9:e1003269 [CrossRef][PubMed]
    [Google Scholar]
  10. Balasubramanian D, Vanderpool CK. Deciphering the interplay between two independent functions of the small RNA regulator SgrS in Salmonella. J Bacteriol 2013;195:4620–4630 [CrossRef][PubMed]
    [Google Scholar]
  11. Shimizu T, Yaguchi H, Ohtani K, Banu S, Hayashi H. Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins. Mol Microbiol 2002;43:257–265 [CrossRef][PubMed]
    [Google Scholar]
  12. Roberts SA, Scott JR. RivR and the small RNA RivX: the missing links between the CovR regulatory cascade and the Mga regulon. Mol Microbiol 2007;66:1506–1522 [CrossRef][PubMed]
    [Google Scholar]
  13. Sonnleitner E, Gonzalez N, Sorger-Domenigg T, Heeb S, Richter AS et al. The small RNA PhrS stimulates synthesis of the Pseudomonas aeruginosa quinolone signal. Mol Microbiol 2011;80:868–885 [CrossRef][PubMed]
    [Google Scholar]
  14. Licht A, Preis S, Brantl S. Implication of CcpN in the regulation of a novel untranslated RNA (SR1) in Bacillus subtilis. Mol Microbiol 2005;58:189–206 [CrossRef][PubMed]
    [Google Scholar]
  15. Heidrich N, Chinali A, Gerth U, Brantl S. The small untranslated RNA SR1 from the B. subtilis genome is involved in the regulation of arginine catabolism. Mol Microbiol 2006;62:520–536 [CrossRef][PubMed]
    [Google Scholar]
  16. Heidrich N, Moll I, Brantl S. In vitro analysis of the interaction between the small RNA SR1 and its primary target ahrC mRNA. Nucleic Acids Res 2007;35:4331–4346 [CrossRef][PubMed]
    [Google Scholar]
  17. Gimpel M, Heidrich N, Mäder U, Krügel H, Brantl S. A dual-function sRNA from Bacillus subtilis: SR1 acts as a peptide encoding mRNA on the gapA operon. Mol Microbiol 2010;76:990–1009 [CrossRef][PubMed]
    [Google Scholar]
  18. Licht A, Brantl S. Transcriptional repressor CcpN from Bacillus subtilis compensates asymmetric contact distribution by cooperative binding. J Mol Biol 2006;364:434–448 [CrossRef][PubMed]
    [Google Scholar]
  19. Licht A, Brantl S. The transcriptional repressor CcpN from Bacillus subtilis uses different repression mechanisms at different promoters. J Biol Chem 2009;284:30032–30038 [CrossRef][PubMed]
    [Google Scholar]
  20. Licht A, Golbik R, Brantl S. Identification of ligands affecting the activity of the transcriptional repressor CcpN from Bacillus subtilis. J Mol Biol 2008;380:17–30 [CrossRef][PubMed]
    [Google Scholar]
  21. Gimpel M, Brantl S. Dual-function sRNA encoded peptide SR1P modulates moonlighting activity of B. subtilis GapA. RNA Biol 2016;13:916–926 [CrossRef][PubMed]
    [Google Scholar]
  22. Gimpel M, Preis H, Barth E, Gramzow L, Brantl S. SR1–a small RNA with two remarkably conserved functions. Nucleic Acids Res 2012;40:11659–11672 [CrossRef][PubMed]
    [Google Scholar]
  23. Didierjean C, Corbier C, Fatih M, Favier F, Boschi-Muller S et al. Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate. J Biol Chem 2003;278:12968–12976 [CrossRef][PubMed]
    [Google Scholar]
  24. Baer R, Bankier AT, Biggin MD, Deininger PL, Farrell PJ et al. DNA sequence and expression of the B95-8 Epstein-Barr virus genome. Nature 1984;310:207–211 [CrossRef][PubMed]
    [Google Scholar]
  25. Gimpel M, Brantl S. Construction of a modular plasmid family for chromosomal integration in Bacillus subtilis. J Microbiol Methods 2012;91:312–317 [CrossRef][PubMed]
    [Google Scholar]
  26. Brantl S, Wagner EG. Dual function of the copR gene product of plasmid pIP501. J Bacteriol 1997;179:7016–7024 [CrossRef][PubMed]
    [Google Scholar]
  27. Preis H, Eckart RA, Gudipati RK, Heidrich N, Brantl S. CodY activates transcription of a small RNA in Bacillus subtilis. J Bacteriol 2009;191:5446–5457 [CrossRef][PubMed]
    [Google Scholar]
  28. Kim DE, Chivian D, Baker D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res 2004;32:W526–W531 [CrossRef][PubMed]
    [Google Scholar]
  29. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 2004;25:1605–1612 [CrossRef][PubMed]
    [Google Scholar]
  30. Böhm G, Muhr R, Jaenicke R. Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Eng 1992;5:191–195 [CrossRef][PubMed]
    [Google Scholar]
  31. Moniot S, Bruno S, Vonrhein C, Didierjean C, Boschi-Muller S et al. Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus. Direct evidence for a flip-flop mechanism. J Biol Chem 2008;283:21693–21702 [CrossRef][PubMed]
    [Google Scholar]
  32. Impens F, Rolhion N, Radoshevich L, Bécavin C, Duval M et al. N-terminomics identifies Prli42 as a membrane miniprotein conserved in Firmicutes and critical for stressosome activation in Listeria monocytogenes. Nat Microbiol 2017;2:17005 [CrossRef][PubMed]
    [Google Scholar]
  33. Commichau FM, Rothe FM, Herzberg C, Wagner E, Hellwig D et al. Novel activities of glycolytic enzymes in Bacillus subtilis: interactions with essential proteins involved in mRNA processing. Mol Cell Proteomics 2009;8:1350–1360 [CrossRef][PubMed]
    [Google Scholar]
  34. Lloyd CR, Park S, Fei J, Vanderpool CK. The small protein SgrT controls transport activity of the glucose-specific phosphotransferase system. J Bacteriol 2017;199:e00869-16 [CrossRef][PubMed]
    [Google Scholar]
  35. Fillinger S, Boschi-Muller S, Azza S, Dervyn E, Branlant G et al. Two glyceraldehyde-3-phosphate dehydrogenases with opposite physiological roles in a nonphotosynthetic bacterium. J Biol Chem 2000;275:14031–14037 [CrossRef][PubMed]
    [Google Scholar]
  36. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbour, NY: Cold Spring Harbor Laboratory Press; 1989
    [Google Scholar]
  37. Geissendörfer M, Hillen W. Regulated expression of heterologous genes in Bacillus subtilis using the Tn10 encoded tet regulatory elements. Appl Microbiol Biotechnol 1990;33:657–663 [CrossRef][PubMed]
    [Google Scholar]
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