Spacing requirements for interactions between the C-terminal domain of the α subunit of Escherichia coli RNA polymerase and the cAMP receptor protein

During transcription initiation at bacterial promoters, the C-terminal domain of the RNA polymerase α subunit (αCTD) can interact with DNA-sequence elements (known as UP elements) and with activator proteins. We have constructed a series of semi-synthetic promoters carrying both an UP element and a consensus DNA-binding site for the Escherichia coli cAMP receptor protein (CRP; a factor that activates transcription by making direct contacts with αCTD). At these promoters, the UP element was located at a variety of distances upstream of the CRP-binding site, which was fixed at position -41.5 bp upstream of the transcript start. At some positions, the UP element caused enhanced promoter activity whereas, at other positions, it had very little effect. In no case was the CRP-dependence of the promoter relieved. DNase I and hydroxyl-radical footprinting were used to study ternary RNA polymerase-CRP-promoter complexes formed at two of the most active of these promoters, and co-operativity between the binding of CRP and purified α subunits was studied. The footprints show that αCTD binds to the UP element as it is displaced upstream but that this displacement does not prevent αCTD from being contacted by CRP. Models to account for this are discussed.