1887

Abstract

Regulation of the gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. analysis predicts the formation of three putative stable stem–loop structures with favourable free energies within the 5′ untranslated region of the message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem–loop mutations diminishing loop stability. Utilizing a series of translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the transcript from degradation. Putative loop 3 contains the ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the promoter that is predicted to hybridize across the 5′ untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants with the antisense RNA gene facilitates translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating expression levels.

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2016-11-23
2021-08-01
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References

  1. Arnold T. E., Yu J., Belasco J. G. 1998; mRNA stabilization by the ompA 5′ untranslated region: two protective elements hinder distinct pathways for mRNA degradation. RNA 4:319–330[PubMed]
    [Google Scholar]
  2. Bechhofer D. H., Dubnau D. 1987; Induced mRNA stability in Bacillus subtilis . Proc Natl Acad Sci U S A 84:498–502 [View Article][PubMed]
    [Google Scholar]
  3. Bellaousov S., Reuter J. S., Seetin M. G., Mathews D. H. 2013; RNAstructure: web servers for RNA secondary structure prediction and analysis. Nucleic Acids Res 41:W471–W474 [View Article][PubMed]
    [Google Scholar]
  4. Bergström S., Robbins K., Koomey J. M., Swanson J. 1986; Piliation control mechanisms in Neisseria gonorrhoeae . Proc Natl Acad Sci U S A 83:3890–3894 [View Article][PubMed]
    [Google Scholar]
  5. Cahoon L. A., Seifert H. S. 2013; Transcription of a cis-acting, noncoding, small RNA is required for pilin antigenic variation in Neisseria gonorrhoeae . PLoS Pathog 9:e1003074 [View Article][PubMed]
    [Google Scholar]
  6. Carrick C. S., Fyfe J. A., Davies J. K. 1997; The normally silent sigma54 promoters upstream of the pilE genes of both Neisseria gonorrhoeae and Neisseria meningitidis are functional when transferred to Pseudomonas aeruginosa . Gene 198:89–97 [View Article][PubMed]
    [Google Scholar]
  7. Deana A., Celesnik H., Belasco J. G. 2008; The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal. Nature 451:355–359 [View Article][PubMed]
    [Google Scholar]
  8. Dietrich M., Munke R., Gottschald M., Ziska E., Boettcher J. P., Mollenkopf H., Friedrich A. 2009; The effect of hfq on global gene expression and virulence in Neisseria gonorrhoeae . FEBS J 276:5507–5520 [View Article][PubMed]
    [Google Scholar]
  9. Fyfe J. A., Carrick C. S., Davies J. K. 1995; The pilE gene of Neisseria gonorrhoeae MS11 is transcribed from a sigma 70 promoter during growth in vitro . J Bacteriol 177:3781–3787[PubMed] [CrossRef]
    [Google Scholar]
  10. Fyfe J. A., Davies J. K. 1998; An AT-rich tract containing an integration host factor-binding domain and two UP-like elements enhances transcription from the pilEp1 promoter of Neisseria gonorrhoeae . J Bacteriol 180:2152–2159[PubMed]
    [Google Scholar]
  11. Georg J., Hess W. R. 2011; cis-Antisense RNA, another level of gene regulation in bacteria. Microbiol Mol Biol Rev 75:286–300 [View Article][PubMed]
    [Google Scholar]
  12. Hill S. A., Samuels D. S., Carlson J. H., Wilson J., Hogan D., Lubke L., Belland R. J. 1997; Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae . Mol Microbiol 23:649–656 [View Article][PubMed]
    [Google Scholar]
  13. Hoe C. H., Raabe C. A., Rozhdestvensky T. S., Tang T. H. 2013; Bacterial sRNAs: regulation in stress. Int J Med Microbiol 303:217–229 [View Article][PubMed]
    [Google Scholar]
  14. Kellogg D. S., Cohen I. R., Norins L. C., Schroeter A. L., Reising G. 1968; Neisseria gonorrhoeae II. Colonial variation and pathogenicity during 35 months in vitro . J Bacteriol 96:596–605[PubMed]
    [Google Scholar]
  15. Koomey M., Bergstrom S., Blake M., Swanson J. 1991; Pilin expression and processing in pilus mutants of Neisseria gonorrhoeae: critical role of Gly-1 in assembly. Mol Microbiol 5:279–287 [View Article][PubMed]
    [Google Scholar]
  16. Marzi S., Fechter P., Chevalier C., Romby P., Geissmann T. 2008; RNA switches regulate initiation of translation in bacteria. Biol Chem 389:585–598 [View Article][PubMed]
    [Google Scholar]
  17. Masters T. L., Wachter S., Wachter J., Hill S. A. 2016; H-NS suppresses pilE intragenic transcription and antigenic variation in Neisseria gonorrhoeae . Microbiology 162:177–190 [View Article][PubMed]
    [Google Scholar]
  18. Pannekoek Y., Huis in 't Veld R., Hopman C. T., Langerak A. A., Speijer D., van der Ende A. 2009; Molecular characterization and identification of proteins regulated by Hfq in Neisseria meningitidis . FEMS Microbiol Lett 294:216–224 [View Article][PubMed]
    [Google Scholar]
  19. Ramsey M. E., Bender T., Klimowicz A. K., Hackett K. T., Yamamoto A., Jolicoeur A., Callaghan M. M., Wassarman K. M., van der Does C., Dillard J. P. 2015; Targeted mutagenesis of intergenic regions in the Neisseria gonorrhoeae gonococcal genetic island reveals multiple regulatory mechanisms controlling type IV secretion. Mol Microbiol 97:1168–1185 [View Article][PubMed]
    [Google Scholar]
  20. Régnier P., Arraiano C. M. 2000; Degradation of mRNA in bacteria: emergence of ubiquitous features. BioEssays 22:235–244 [View Article][PubMed]
    [Google Scholar]
  21. Schuck A., Diwa A., Belasco J. G. 2009; RNase E autoregulates its synthesis in Escherichia coli by binding directly to a stem-loop in the rne 5′ untranslated region. Mol Microbiol 72:470–478 [View Article][PubMed]
    [Google Scholar]
  22. Sittka A., Pfeiffer V., Tedin K., Vogel J. 2007; The RNA chaperone Hfq is essential for the virulence of Salmonella typhimurium . Mol Microbiol 63:193–217 [View Article][PubMed]
    [Google Scholar]
  23. Swanson J. 1973; Studies on gonococcus infection. IV. Pili: their role in attachment of gonococci to tissue culture cells. J Exp Med 127:571–589 [CrossRef]
    [Google Scholar]
  24. Swanson J. 1982; Colony opacity and protein II compositions of gonococci. Infect Immun 37:359–368[PubMed]
    [Google Scholar]
  25. Swanson J., Robbins K., Barrera O., Corwin D., Boslego J., Ciak J., Blake M., Koomey J. M. 1987; Gonococcal pilin variants in experimental gonorrhea. J Exp Med 165:1344–1357 [View Article][PubMed]
    [Google Scholar]
  26. Tønjum T., Koomey M. 1997; The pilus colonization factor of pathogenic neisserial species: organelle biogenesis and structure/function relationships – a review. Gene 192:155–163 [View Article][PubMed]
    [Google Scholar]
  27. Uppal S., Akkipeddi V. S., Jawali N. 2008; Posttranscriptional regulation of cspE in Escherichia coli: involvement of the short 5′-untranslated region. FEMS Microbiol Lett 279:83–91 [View Article][PubMed]
    [Google Scholar]
  28. Wachter J., Hill S. A. 2015; Small transcriptome analysis indicates that the enzyme RppH influences both the quality and quantity of sRNAs in Neisseria gonorrhoeae . FEMS Microbiol Lett 362:1–7 [View Article]
    [Google Scholar]
  29. Wachter J., Masters T. L., Wachter S., Mason J., Hill S. A. 2015; pilS loci in Neisseria gonorrhoeae are transcriptionally active. Microbiology 161:1124–1135 [View Article][PubMed]
    [Google Scholar]
  30. Wade J. T., Grainger D. C. 2014; Pervasive transcription: illuminating the dark matter of bacterial transcriptomes. Nat Rev Microbiol 12:647–653 [View Article][PubMed]
    [Google Scholar]
  31. Waters L. S., Storz G. 2009; Regulatory RNAs in bacteria. Cell 136:615–628 [View Article][PubMed]
    [Google Scholar]
  32. Zuker M. 2003; Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415 [View Article][PubMed]
    [Google Scholar]
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