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

Research output: Contribution to journalArticle

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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 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
Volume179
Issue number5
DOIs
StatePublished - Jan 1 1997

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Bordetella pertussis
Hemagglutinins
DNA-Directed DNA Polymerase
DNA-Directed RNA Polymerases
Phosphates
Escherichia coli
Escherichia coli Proteins
DNA
Pertussis Toxin
Virulence Factors
Mutant Proteins
Occupations
Alanine
Signal Transduction
Nucleotides
Enzymes
Genes
In Vitro Techniques

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology

Cite this

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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 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.",
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Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter. / Boucher, Philip E.; Murakami, Katsuhiko; Ishihama, Akira; Stibitz, Scott.

In: Journal of bacteriology, Vol. 179, No. 5, 01.01.1997, p. 1755-1763.

Research output: Contribution to journalArticle

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T1 - Nature of DNA binding and RNA polymerase interaction of the Bordetella pertussis BvgA transcriptional activator at the fha promoter

AU - Boucher, Philip E.

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AU - Stibitz, Scott

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