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

Philip E. Boucher, Katsuhiko Murakami, Akira Ishihama, Scott Stibitz

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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 α 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 attire.

Original languageEnglish (US)
Pages (from-to)1755-1763
Number of pages9
JournalJournal of bacteriology
Issue number5
StatePublished - 1997

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology


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