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. 1997 Mar;179(5):1755–1763. doi: 10.1128/jb.179.5.1755-1763.1997

Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter.

P E Boucher 1, K Murakami 1, A Ishihama 1, S Stibitz 1
PMCID: PMC178891  PMID: 9045838

Abstract

The expression of virulence factor genes in Bordetella pertussis is mediated by the BvgA-BvgS two-component signal transduction system. The response regulator, BvgA, acts directly as a transcriptional activator at the loci encoding pertussis toxin (ptx) and filamentous hemagglutinin (fha). Previous studies have demonstrated that these two loci are differentially regulated by BvgA. As an initial step in gaining insight into the mechanism underlying this differential regulation, we initiated DNA binding and in vitro transcription analyses to examine the activities of BvgA and RNA polymerase (RNAP) purified from both B. pertussis and Escherichia coli at the fha promoter. We discovered that unphosphorylated BvgA binds to a single region (-100 to -70, relative to the start of transcription), whereas phosphorylated BvgA binds both this region and another, farther downstream, that extends to the -35 nucleotide. In the absence of BvgA, RNAP binds a region farther upstream than expected (-104 to -35). However, occupation of both sites by BvgA phosphate repositions RNAP to the site used in vivo. The binding of BvgA phosphate to the downstream site correlates with in vitro transcriptional activity at the fha promoter. As the DNA binding and transcription activities of the E. coli-derived RNAP are similar to those observed for the B. pertussis enzyme, we employed several mutant E. coli proteins in in vitro transcription analyses. We observed that polymerases carrying either a deletion of the C-terminal domain of the alpha subunit or substitution of alanine at either of two critical residues within this domain were severely impaired in the ability to mediate BvgA-activated transcription at fha.

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Selected References

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  1. Ansari A. Z., Bradner J. E., O'Halloran T. V. DNA-bend modulation in a repressor-to-activator switching mechanism. Nature. 1995 Mar 23;374(6520):371–375. doi: 10.1038/374370a0. [DOI] [PubMed] [Google Scholar]
  2. Aricó B., Miller J. F., Roy C., Stibitz S., Monack D., Falkow S., Gross R., Rappuoli R. Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6671–6675. doi: 10.1073/pnas.86.17.6671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boucher P. E., Menozzi F. D., Locht C. The modular architecture of bacterial response regulators. Insights into the activation mechanism of the BvgA transactivator of Bordetella pertussis. J Mol Biol. 1994 Aug 19;241(3):363–377. doi: 10.1006/jmbi.1994.1513. [DOI] [PubMed] [Google Scholar]
  4. Boucher P. E., Stibitz S. Synergistic binding of RNA polymerase and BvgA phosphate to the pertussis toxin promoter of Bordetella pertussis. J Bacteriol. 1995 Nov;177(22):6486–6491. doi: 10.1128/jb.177.22.6486-6491.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carbonetti N. H., Fuchs T. M., Patamawenu A. A., Irish T. J., Deppisch H., Gross R. Effect of mutations causing overexpression of RNA polymerase alpha subunit on regulation of virulence factors in Bordetella pertussis. J Bacteriol. 1994 Dec;176(23):7267–7273. doi: 10.1128/jb.176.23.7267-7273.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Choi S. H., Greenberg E. P. Genetic dissection of DNA binding and luminescence gene activation by the Vibrio fischeri LuxR protein. J Bacteriol. 1992 Jun;174(12):4064–4069. doi: 10.1128/jb.174.12.4064-4069.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cunningham B. C., Wells J. A. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. 1989 Jun 2;244(4908):1081–1085. doi: 10.1126/science.2471267. [DOI] [PubMed] [Google Scholar]
  8. Danot O., Raibaud O. Multiple protein-DNA and protein-protein interactions are involved in transcriptional activation by MalT. Mol Microbiol. 1994 Oct;14(2):335–346. doi: 10.1111/j.1365-2958.1994.tb01294.x. [DOI] [PubMed] [Google Scholar]
  9. Drew H. R., Travers A. A. DNA bending and its relation to nucleosome positioning. J Mol Biol. 1985 Dec 20;186(4):773–790. doi: 10.1016/0022-2836(85)90396-1. [DOI] [PubMed] [Google Scholar]
  10. Elliott T., Geiduschek E. P. Defining a bacteriophage T4 late promoter: absence of a "-35" region. Cell. 1984 Jan;36(1):211–219. doi: 10.1016/0092-8674(84)90091-6. [DOI] [PubMed] [Google Scholar]
  11. Goff C. G. Coliphage-induced ADP-ribosylation of Escherichia coli RNA polymerase. Methods Enzymol. 1984;106:418–429. doi: 10.1016/0076-6879(84)06045-6. [DOI] [PubMed] [Google Scholar]
  12. Hochschild A., Ptashne M. Cooperative binding of lambda repressors to sites separated by integral turns of the DNA helix. Cell. 1986 Mar 14;44(5):681–687. doi: 10.1016/0092-8674(86)90833-0. [DOI] [PubMed] [Google Scholar]
  13. Ishihama A. Protein-protein communication within the transcription apparatus. J Bacteriol. 1993 May;175(9):2483–2489. doi: 10.1128/jb.175.9.2483-2489.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Ishihama A. Role of the RNA polymerase alpha subunit in transcription activation. Mol Microbiol. 1992 Nov;6(22):3283–3288. doi: 10.1111/j.1365-2958.1992.tb02196.x. [DOI] [PubMed] [Google Scholar]
  15. Jeon Y. H., Negishi T., Shirakawa M., Yamazaki T., Fujita N., Ishihama A., Kyogoku Y. Solution structure of the activator contact domain of the RNA polymerase alpha subunit. Science. 1995 Dec 1;270(5241):1495–1497. doi: 10.1126/science.270.5241.1495. [DOI] [PubMed] [Google Scholar]
  16. Karimova G., Bellalou J., Ullmann A. Phosphorylation-dependent binding of BvgA to the upstream region of the cyaA gene of Bordetella pertussis. Mol Microbiol. 1996 May;20(3):489–496. doi: 10.1046/j.1365-2958.1996.5231057.x. [DOI] [PubMed] [Google Scholar]
  17. Kolb A., Busby S., Buc H., Garges S., Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. doi: 10.1146/annurev.bi.62.070193.003533. [DOI] [PubMed] [Google Scholar]
  18. Merkel T. J., Stibitz S. Identification of a locus required for the regulation of bvg-repressed genes in Bordetella pertussis. J Bacteriol. 1995 May;177(10):2727–2736. doi: 10.1128/jb.177.10.2727-2736.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miller J. F., Roy C. R., Falkow S. Analysis of Bordetella pertussis virulence gene regulation by use of transcriptional fusions in Escherichia coli. J Bacteriol. 1989 Nov;171(11):6345–6348. doi: 10.1128/jb.171.11.6345-6348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Murakami K., Fujita N., Ishihama A. Transcription factor recognition surface on the RNA polymerase alpha subunit is involved in contact with the DNA enhancer element. EMBO J. 1996 Aug 15;15(16):4358–4367. [PMC free article] [PubMed] [Google Scholar]
  21. Muskhelishvili G., Travers A. A., Heumann H., Kahmann R. FIS and RNA polymerase holoenzyme form a specific nucleoprotein complex at a stable RNA promoter. EMBO J. 1995 Apr 3;14(7):1446–1452. doi: 10.1002/j.1460-2075.1995.tb07131.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pérez-Martín J., Rojo F., de Lorenzo V. Promoters responsive to DNA bending: a common theme in prokaryotic gene expression. Microbiol Rev. 1994 Jun;58(2):268–290. doi: 10.1128/mr.58.2.268-290.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rao L., Ross W., Appleman J. A., Gaal T., Leirmo S., Schlax P. J., Record M. T., Jr, Gourse R. L. Factor independent activation of rrnB P1. An "extended" promoter with an upstream element that dramatically increases promoter strength. J Mol Biol. 1994 Feb 4;235(5):1421–1435. doi: 10.1006/jmbi.1994.1098. [DOI] [PubMed] [Google Scholar]
  24. Raumann B. E., Rould M. A., Pabo C. O., Sauer R. T. DNA recognition by beta-sheets in the Arc repressor-operator crystal structure. Nature. 1994 Feb 24;367(6465):754–757. doi: 10.1038/367754a0. [DOI] [PubMed] [Google Scholar]
  25. Ross W., Gosink K. K., Salomon J., Igarashi K., Zou C., Ishihama A., Severinov K., Gourse R. L. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993 Nov 26;262(5138):1407–1413. doi: 10.1126/science.8248780. [DOI] [PubMed] [Google Scholar]
  26. Roy C. R., Falkow S. Identification of Bordetella pertussis regulatory sequences required for transcriptional activation of the fhaB gene and autoregulation of the bvgAS operon. J Bacteriol. 1991 Apr;173(7):2385–2392. doi: 10.1128/jb.173.7.2385-2392.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Roy C. R., Miller J. F., Falkow S. The bvgA gene of Bordetella pertussis encodes a transcriptional activator required for coordinate regulation of several virulence genes. J Bacteriol. 1989 Nov;171(11):6338–6344. doi: 10.1128/jb.171.11.6338-6344.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Scarlato V., Aricò B., Prugnola A., Rappuoli R. Sequential activation and environmental regulation of virulence genes in Bordetella pertussis. EMBO J. 1991 Dec;10(12):3971–3975. doi: 10.1002/j.1460-2075.1991.tb04967.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Scarlato V., Prugnola A., Aricó B., Rappuoli R. Positive transcriptional feedback at the bvg locus controls expression of virulence factors in Bordetella pertussis. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6753–6757. doi: 10.1073/pnas.87.17.6753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Steffen P., Goyard S., Ullmann A. Phosphorylated BvgA is sufficient for transcriptional activation of virulence-regulated genes in Bordetella pertussis. EMBO J. 1996 Jan 2;15(1):102–109. [PMC free article] [PubMed] [Google Scholar]
  32. Stibitz S., Aaronson W., Monack D., Falkow S. Phase variation in Bordetella pertussis by frameshift mutation in a gene for a novel two-component system. Nature. 1989 Mar 16;338(6212):266–269. doi: 10.1038/338266a0. [DOI] [PubMed] [Google Scholar]
  33. Stibitz S. Mutations in the bvgA gene of Bordetella pertussis that differentially affect regulation of virulence determinants. J Bacteriol. 1994 Sep;176(18):5615–5621. doi: 10.1128/jb.176.18.5615-5621.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stibitz S., Weiss A. A., Falkow S. Genetic analysis of a region of the Bordetella pertussis chromosome encoding filamentous hemagglutinin and the pleiotropic regulatory locus vir. J Bacteriol. 1988 Jul;170(7):2904–2913. doi: 10.1128/jb.170.7.2904-2913.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tang H., Severinov K., Goldfarb A., Fenyo D., Chait B., Ebright R. H. Location, structure, and function of the target of a transcriptional activator protein. Genes Dev. 1994 Dec 15;8(24):3058–3067. doi: 10.1101/gad.8.24.3058. [DOI] [PubMed] [Google Scholar]
  36. Tao K., Fujita N., Ishihama A. Involvement of the RNA polymerase alpha subunit C-terminal region in co-operative interaction and transcriptional activation with OxyR protein. Mol Microbiol. 1993 Mar;7(6):859–864. doi: 10.1111/j.1365-2958.1993.tb01176.x. [DOI] [PubMed] [Google Scholar]
  37. Tao K., Zou C., Fujita N., Ishihama A. Mapping of the OxyR protein contact site in the C-terminal region of RNA polymerase alpha subunit. J Bacteriol. 1995 Dec;177(23):6740–6744. doi: 10.1128/jb.177.23.6740-6744.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Thompson N. E., Hager D. A., Burgess R. R. Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase. Biochemistry. 1992 Aug 4;31(30):7003–7008. doi: 10.1021/bi00145a019. [DOI] [PubMed] [Google Scholar]
  39. Uhl M. A., Miller J. F. Autophosphorylation and phosphotransfer in the Bordetella pertussis BvgAS signal transduction cascade. Proc Natl Acad Sci U S A. 1994 Feb 1;91(3):1163–1167. doi: 10.1073/pnas.91.3.1163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wang Q., Calvo J. M. Lrp, a major regulatory protein in Escherichia coli, bends DNA and can organize the assembly of a higher-order nucleoprotein structure. EMBO J. 1993 Jun;12(6):2495–2501. doi: 10.1002/j.1460-2075.1993.tb05904.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Weiss A. A., Hewlett E. L., Myers G. A., Falkow S. Tn5-induced mutations affecting virulence factors of Bordetella pertussis. Infect Immun. 1983 Oct;42(1):33–41. doi: 10.1128/iai.42.1.33-41.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wells J. A. Systematic mutational analyses of protein-protein interfaces. Methods Enzymol. 1991;202:390–411. doi: 10.1016/0076-6879(91)02020-a. [DOI] [PubMed] [Google Scholar]

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