1887

Abstract

6S RNA is a small, non-coding RNA that interacts directly with -RNA polymerase and regulates transcription at many -dependent promoters. Here, we demonstrate that 6S RNA regulates transcription of , which encodes a ppGpp synthase. The 6S RNA-dependent regulation of expression results in increased ppGpp levels during early stationary phase in cells lacking 6S RNA. These changes in ppGpp levels, although modest, are sufficient to result in altered regulation of transcription from -dependent promoters sensitive to ppGpp, including those promoting expression of genes involved in amino acid biosynthesis and rRNA. These data place 6S RNA as another player in maintaining appropriate gene expression as cells transition into stationary phase. Independent of this ppGpp-mediated 6S RNA-dependent regulation, we also demonstrate that in later stationary phase, 6S RNA continues to downregulate transcription in general, and specifically at a subset of the amino acid promoters, but through a mechanism that is independent of ppGpp and which we hypothesize is through direct regulation. In addition, 6S RNA-dependent regulation of activity is not mediated through observed changes in ppGpp levels. We suggest a role for 6S RNA in modulating transcription of several global regulators directly, including , to downregulate expression of key pathways in response to changing environmental conditions.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.043992-0
2010-12-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/156/12/3791.html?itemId=/content/journal/micro/10.1099/mic.0.043992-0&mimeType=html&fmt=ahah

References

  1. Barker, M. M., Gaal, T., Josaitis, C. A. & Gourse, R. L. ( 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.[CrossRef]
    [Google Scholar]
  2. Bernardo, L. M., Johansson, L. U., Solera, D., Skärfstad, E. & Shingler, V. ( 2006; ). The guanosine tetraphosphate (ppGpp) alarmone, DksA and promoter affinity for RNA polymerase in regulation of σ 54-dependent transcription. Mol Microbiol 60, 749–764.[CrossRef]
    [Google Scholar]
  3. Bown, J. A., Barne, K. A., Minchin, S. D. & Busby, S. J. W. ( 1997; ). Extended −10 promoters. Nucleic Acids Mol Biol 11, 41–52.
    [Google Scholar]
  4. Cavanagh, A. T., Klocko, A. D., Liu, X. & Wassarman, K. M. ( 2008; ). Promoter specificity for 6S RNA regulation of transcription is determined by core promoter sequences and competition for region 4.2 of σ 70. Mol Microbiol 67, 1242–1256.[CrossRef]
    [Google Scholar]
  5. Costanzo, A., Nicoloff, H., Barchinger, S. E., Banta, A. B., Gourse, R. L. & Ades, S. E. ( 2008; ). ppGpp and DksA likely regulate the activity of the extracytoplasmic stress factor σ E in Escherichia coli by both direct and indirect mechanisms. Mol Microbiol 67, 619–632.[CrossRef]
    [Google Scholar]
  6. Darwin, A. J. ( 2005; ). The phage-shock-protein response. Mol Microbiol 57, 621–628.[CrossRef]
    [Google Scholar]
  7. Gaal, T., Ross, W., Estrem, S. T., Nguyen, L. H., Burgess, R. R. & Gourse, R. L. ( 2001; ). Promoter recognition and discrimination by Eσ S RNA polymerase. Mol Microbiol 42, 939–954.[CrossRef]
    [Google Scholar]
  8. Gralla, J. D. ( 2005; ). Escherichia coli ribosomal RNA transcription: regulatory roles for ppGpp, NTPs, architectural proteins and a polymerase-binding protein. Mol Microbiol 55, 973–977.
    [Google Scholar]
  9. Hindley, J. ( 1967; ). Fractionation of 32P-labelled ribonucleic acids on polyacrylamide gels and their characterization by fingerprinting. J Mol Biol 30, 125–136.[CrossRef]
    [Google Scholar]
  10. Hirvonen, C. A., Ross, W., Wozniak, C. E., Marasco, E., Anthony, J. R., Aiyar, S. E., Newburn, V. H. & Gourse, R. L. ( 2001; ). Contributions of UP elements and the transcription factor FIS to expression from the seven rrn P1 promoters in Escherichia coli. J Bacteriol 183, 6305–6314.[CrossRef]
    [Google Scholar]
  11. Jishage, M., Kvint, K., Shingler, V. & Nyström, T. ( 2002; ). Regulation of sigma factor competition by the alarmone ppGpp. Genes Dev 16, 1260–1270.[CrossRef]
    [Google Scholar]
  12. Klocko, A. D. & Wassarman, K. M. ( 2009; ). 6S RNA binding to Eσ 70 requires a positively charged surface of σ 70 region 4.2. Mol Microbiol 73, 152–164.[CrossRef]
    [Google Scholar]
  13. Lee, C. A., Fournier, M. J. & Beckwith, J. ( 1985; ). Escherichia coli 6S RNA is not essential for growth or protein secretion. J Bacteriol 161, 1156–1161.
    [Google Scholar]
  14. Magnusson, L. U., Farewell, A. & Nyström, T. ( 2005; ). ppGpp: a global regulator in Escherichia coli. Trends Microbiol 13, 236–242.[CrossRef]
    [Google Scholar]
  15. Metzger, S., Dror, I. B., Aizenman, E., Schreiber, G., Toone, M., Friesen, J. D., Cashel, M. & Glaser, G. ( 1988; ). The nucleotide sequence and characterization of the relA gene of Escherichia coli. J Biol Chem 263, 15699–15704.
    [Google Scholar]
  16. Miller, J. H. ( 1972; ). Experiments in Molecular Genetics. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  17. Mitchell, J. E., Zheng, D., Busby, S. J. W. & Minchin, S. D. ( 2003; ). Identification and analysis of “extended –10” promoters in Escherichia coli. Nucleic Acids Res 31, 4689–4695.[CrossRef]
    [Google Scholar]
  18. Model, P., Jovanovic, G. & Dworkin, J. ( 1997; ). The Escherichia coli phage-shock-protein (psp) operon. Mol Microbiol 24, 255–261.[CrossRef]
    [Google Scholar]
  19. Murray, H. D., Schneider, D. A. & Gourse, R. L. ( 2003; ). Control of rRNA expression by small molecules is dynamic and nonredundant. Mol Cell 12, 125–134.[CrossRef]
    [Google Scholar]
  20. Nakagawa, A., Oshima, T. & Mori, H. ( 2006; ). Identification and characterization of a second, inducible promoter of relA in Escherichia coli. Genes Genet Syst 81, 299–310.[CrossRef]
    [Google Scholar]
  21. Neidhardt, F. C., Bloch, P. L. & Smith, D. F. ( 1974; ). Culture medium for enterobacteria. J Bacteriol 119, 736–747.
    [Google Scholar]
  22. Neusser, T., Polen, T., Geissen, R. & Wagner, R. ( 2010; ). Depletion of the non-coding regulatory 6S RNA in E. coli causes a surprising reduction in the expression of the translation machinery. BMC Genomics 11, 165–178.[CrossRef]
    [Google Scholar]
  23. Paul, B. J., Ross, W., Gaal, T. & Gourse, R. L. ( 2004; ). rRNA transcription in Escherichia coli. Annu Rev Genet 38, 749–770.[CrossRef]
    [Google Scholar]
  24. Potrykus, K. & Cashel, M. ( 2008; ). (p)ppGpp: still magical? Annu Rev Microbiol 62, 35–51.[CrossRef]
    [Google Scholar]
  25. Rao, L., Ross, W., Appleman, J. A., Gaal, T., Leirmo, S., Schlax, P. J., Record, M. T., Jr & Gourse, R. L. ( 1994; ). Factor independent activation of rrnBP1. An “extended” promoter with an upstream element that dramatically increases promoter strength. J Mol Biol 235, 1421–1435.[CrossRef]
    [Google Scholar]
  26. Schneider, D. A. & Gourse, R. L. ( 2004; ). Relationship between growth rate and ATP concentration in Escherichia coli: a bioassay for available cellular ATP. J Biol Chem 279, 8262–8268.[CrossRef]
    [Google Scholar]
  27. Schneider, D. A., Murray, H. D. & Gourse, R. L. ( 2003; ). Measuring control of transcription initiation by changing concentrations of nucleotides and their derivatives. Methods Enzymol 370, 606–617.
    [Google Scholar]
  28. Silhavy, T. J., Berman, M. L. & Enquist, L. W. ( 1984; ). Experiments with Gene Fusions. Cold Spring Harbor, NY. : Cold Spring Harbor Laboratory.
    [Google Scholar]
  29. Srivatsan, A. & Wang, J. D. ( 2008; ). Control of bacterial transcription, translation and replication by (p)ppGpp. Curr Opin Microbiol 11, 100–105.[CrossRef]
    [Google Scholar]
  30. Trotochaud, A. E. & Wassarman, K. M. ( 2004; ). 6S RNA function enhances long-term cell survival. J Bacteriol 186, 4978–4985.[CrossRef]
    [Google Scholar]
  31. Trotochaud, A. E. & Wassarman, K. M. ( 2005; ). A highly conserved 6S RNA structure is required for regulation of transcription. Nat Struct Mol Biol 12, 313–319.[CrossRef]
    [Google Scholar]
  32. Trotochaud, A. E. & Wassarman, K. M. ( 2006; ). 6S RNA regulation of pspF transcription leads to altered cell survival at high pH. J Bacteriol 188, 3936–3943.[CrossRef]
    [Google Scholar]
  33. Voskuil, M. I., Voepel, K. & Chambliss, G. H. ( 1995; ). The −16 region, a vital sequence for the utilization of a promoter in Bacillus subtilis and Escherichia coli. Mol Microbiol 17, 271–279.[CrossRef]
    [Google Scholar]
  34. Wassarman, K. M. ( 2007; ). 6S RNA: a regulator of transcription. Mol Microbiol 65, 1425–1431.[CrossRef]
    [Google Scholar]
  35. Wassarman, K. M. & Saecker, R. M. ( 2006; ). Synthesis-mediated release of a small RNA inhibitor of RNA polymerase. Science 314, 1601–1603.[CrossRef]
    [Google Scholar]
  36. Wassarman, K. M. & Storz, G. ( 2000; ). 6S RNA regulates E. coli RNA polymerase activity. Cell 101, 613–623.[CrossRef]
    [Google Scholar]
  37. Willkomm, D. K. & Hartmann, R. K. ( 2005; ). 6S RNA – an ancient regulator of bacterial RNA polymerase rediscovered. Biol Chem 386, 1273–1277.
    [Google Scholar]
  38. Xiao, H., Kalman, M., Ikehara, K., Zemel, S., Glaser, G. & Cashel, M. ( 1991; ). Residual guanosine 3′,5′-bispyrophosphate synthetic activity of relA null mutants can be eliminated by spoT null mutations. J Biol Chem 266, 5980–5990.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.043992-0
Loading
/content/journal/micro/10.1099/mic.0.043992-0
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error