The essential role of the promoter-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcription by CAP from -215.
Catabolite gene activator protein (CAP) is essential for the expression of Pap pili by uropathogenic Escherichia coli. Both in vitro and in vivo analyses indicate that binding of cAMP-CAP centred at 215.5 bp upstream of the papBA promoter is essential for activation of transcription. CAP-dependent activation of papBA requires binding of the leucine-responsive regulatory protein (Lrp) at binding sites that extend from -180 to -149 relative to the start site of papBA. Our data indicate that CAP and Lrp bind independently to their respective pap DNA sites. Activation of papBA transcription was eliminated by mutations in the activating region 1 (AR1) of CAP, but not in the AR2 region, similar to class I CAP-dependent promoters. Also, like class I promoters, the C-terminal domain of the alpha-subunit of RNA polymerase appears to play a role in transcription activation. Moreover, phase variation is strictly dependent upon the helical phase of the CAP DNA binding site with respect to the papBA transcription start site. Using an 'oriented heterodimer' approach with wild-type and AR1 mutant CAPs, it was shown that the AR1 region of the CAP subunit proximal to papBA is required for stimulation of papBA transcription, whereas AR1 of the promoter-distal subunit is not. Previously, CAP was hypothesized to activate pap transcription indirectly by disrupting repression mediated by H-NS. The results presented here show that AR1 of the promoter-proximal CAP subunit was required for papBA transcription even in the absence of the histone-like protein H-NS. These results show that the promoter-proximal subunit of CAP, bound 215.5 bp upstream of the papBA transcription start site, plays an active role in stimulating papBA transcription, possibly by interacting with the C-terminal domain of the alpha-subunit of RNA polymerase.
Weyand
N J
NJ
Department of Molecular, Cellular and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA 93106, USA.
Braaten
B A
BA
van der Woude
M
M
Tucker
J
J
Low
D A
DA
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D002373:Cyclic AMP Receptor Protein / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D036781:Fimbriae Proteins; D051756:Leucine-Responsive Regulatory Protein; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D021122:Protein Subunits; D012045:Regulatory Sequences, Nucleic Acid; D012097:Repressor Proteins; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
C078802:AtpA protein, E coli; D001426:Bacterial Proteins; D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C497278:Lrp protein, E coli; D008565:Membrane Proteins; C086707:PapI protein, E coli; D021122:Protein Subunits; D012097:Repressor Proteins; D014157:Transcription Factors; C116245:atpD protein, E coli; D051756:Leucine-Responsive Regulatory Protein; D036781:Fimbriae Proteins; D012321:DNA-Directed RNA Polymerases; C113275:RNA polymerase alpha subunit