CAP, the ÿ45 Region, and RNA Polymerase: Three Partners in Transcription Initiation at lacP1 in Escherichia coli
نویسندگان
چکیده
0022±2836/98/380495±10 $30.00/0 The lac operon of Escherichia coli is positively regulated by the catabolite activator protein (CAP) bound upstream of the ÿ45 region (CAP binding is centered at ÿ61.5; the ÿ45 region extends from ÿ50 to ÿ38). Certain mutations within the ÿ45 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 ÿ45 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 ÿ45 region UP element against a heparin challenge. This shows that CAP and the UP DNA do not compete for the a-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 twoto threefold and occurs both in vivo and in vitro. It is not dependent upon the a-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This twoto threefold invariable CAP stimulation appears to depend on the ÿ45 region sequence as our ÿ45 region mutants demonstrate different responses to HL159 CAP stimulation in vivo. # 1998 Academic Press
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