CAP, the −45 region, and RNA polymerase: three partners in transcription initiation at lacP1 in Escherichia coli

Abstract

The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the −45 region (CAP binding is centered at −61.5; the −45 region extends from −50 to −38). Certain mutations within the −45 region generate sequences that resemble UP elements in base composition and mimic the stimulation by the rrnBP1 UP element, yielding up to 15-fold stimulation in vivo. These −45 region “UP mutants” are compromised in their CAP stimulation. CAP and UP elements do not act in a fully additive manner in vivo at the lac operon. Transcription assays with the wild-type lac promoter and an UP mutant of lac indicate that CAP and UP DNA also fail to act in a completely additive manner in vitro. RNA polymerase can stabilize CAP binding to promoter DNA with a −45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the α-CTD as a mechanism for their lack of additivity. CAP and UP elements both demonstrate decreased stimulation of transcription as RNA polymerase concentration is increased from 0.05 to 10 nM in in vitro transcription experiments. In addition CAP also stimulates transcription in a manner that does not decrease as RNA polymerase is varied over this concentration range. This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro. It is not dependent upon the α-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two- to threefold invariable CAP stimulation appears to depend on the −45 region sequence as our −45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo.