Scientific Publications by FDA Staff

J Mol Biol 2011 Jun 24;409(5):692-709

Different Requirements for sigma Region 4 in BvgA Activation of the Bordetella pertussis Promoters P(fim3) and P(fhaB).

Decker KB, Chen Q, Hsieh ML, Boucher P, Stibitz S, Hinton DM

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 sigma subunit of RNA polymerase (RNAP). We demonstrate that sigma 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 sigma 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 sigmaD581C-FeBABE positions Region 4 near the -35 region of P(fim3); cleavage using RNAP with alpha276C-FeBABE or alpha302C-FeBABE also positions an alpha 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 alpha subunit C-terminal domain, and sigma Region 4. Molecular modeling suggests how BvgA, sigma Region 4, and alpha might coexist within this DNA in a conformation that suggests a novel mechanism of activation.