title: Different requirements for σ Region 4 in BvgA activation of the Bordetella pertussis promoters P(fim3) and P(fhaB).
abstract: Bordetella pertussis BvgA is a global response regulator that activates virulence genes, including adhesin-encoding fim3 and fhaB. At the fhaB promoter, P(fhaB), a BvgA binding site lies immediately upstream of the -35 promoter element recognized by Region 4 of the σ subunit of RNA polymerase (RNAP). We demonstrate that σ Region 4 is required for BvgA activation of P(fhaB), a hallmark of Class II activation. In contrast, the promoter-proximal BvgA binding site at P(fim3) includes the -35 region, which is composed of a tract of cytosines that lacks specific sequence information. We demonstrate that σ Region 4 is not required for BvgA activation at P(fim3). Nonetheless, Region 4 mutations that impair its typical interactions with core and with the -35 DNA affect P(fim3) transcription. Hydroxyl radical cleavage using RNAP with σD581C-FeBABE positions Region 4 near the -35 region of P(fim3); cleavage using RNAP with α276C-FeBABE or α302C-FeBABE also positions an α subunit C-terminal domain within the -35 region, on a different helical face from the promoter-proximal BvgA~P dimer. Our results suggest that the -35 region of P(fim3) accommodates a BvgA~P dimer, an α subunit C-terminal domain, and σ Region 4. Molecular modeling suggests how BvgA, σ Region 4, and α might coexist within this DNA in a conformation that suggests a novel mechanism of activation.
authors.affiliation: Gene Expression and Regulation Section, Laboratory of Molecular and Cellular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Kimberly B
authors.initials: KB
authors.lastname: Decker
authors.forename: Qing
authors.initials: Q
authors.lastname: Chen
authors.forename: Meng-Lun
authors.initials: ML
authors.lastname: Hsieh
authors.forename: Philip
authors.initials: P
authors.lastname: Boucher
authors.forename: Scott
authors.initials: S
authors.lastname: Stibitz
authors.forename: Deborah M
authors.initials: DM
authors.lastname: Hinton
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D001886:Bordetella pertussis / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; C065601:BvgA protein, Bacteria; D004269:DNA, Bacterial; D014157:Transcription Factors; D017665:Hydroxyl Radical; D012321:DNA-Directed RNA Polymerases