The essential role of the promoter-proximal subunit of CAP in pap phase variation: Lrp- and helical phase-dependent activation of papBA transcriptionby 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 analy ses indicate that binding of cAMP-CAP centred at 215.5 bp upstream of the p apBA promoter is essential for activation of transcription. CAP-dependent a ctivation of papBA requires binding of the leucine-responsive regulatory pr otein (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 el iminated by mutations in the activating region 1 (AR1) of CAP, but not in t he 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 variat ion is strictly dependent upon the helical phase of the CAP DNA binding sit e with respect to the papBA transcription start site. Using an 'oriented he terodimer' approach with wild-type and AR1 mutant CAPs, it was shown that t he AR1 region of the CAP subunit proximal to papBA is required for stimulat ion of papBA transcription, whereas AR1 of the promoter-distal subunit is n ot. Previously, CAP was hypothesized to activate pap transcription indirect ly by disrupting repression mediated by H-NS. The results presented here sh ow that AR1 of the promoter-proximal CAP subunit was required for papBA tra nscription even in the absence of the histone-like protein H-NS. These resu lts 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.