CAP, THE -45 REGION, AND RNA-POLYMERASE - 3 PARTNERS IN TRANSCRIPTIONINITIATION AT LACP1 IN ESCHERICHIA-COLI

The lac operon of Escherichia coli is positively regulated by the cata bolite activator protein (CAP) bound upstream of the -45 region (CAP b inding is centered at -61.5; the -45 region extends from -50 to -38). Certain mutations within the -45 region generate sequences that resemb le UP elements in base composition and mimic the stimulation by the rr nBP1 UP element, yielding up to 15-fold stimulation in vivo. These -45 region ''UP mutants'' are compromised in their CAP stimulation. CAP a nd UP elements do not act in a fully additive manner in vivo at the la c operon. Transcription assays with the wild-type lac promoter and an Ur mutant of Inc indicate that CAP and UP DNA also fail to act in a co mpletely additive manner in vitro. RNA polymerase can stabilize CAP bi nding to promoter DNA with a -45 region UP element against a heparin c hallenge. 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 UT elements b oth demonstrate decreased stimulation of transcription as RNA polymera se concentration is increased from 0.05 to 10 nM in in vitro transcrip tion experiments. In addition CAP also stimulates transcription in a m anner that does not decrease as RNA polymerase is varied over this con centration range. This invariable stimulation is by two- to threefold and occurs both in vivo and in vitro. It is not dependent upon the alp ha-CTD of RNA polymerase and is maintained in the presence of the AR1 CAP mutant HL159. This two- to threefold in invariable CAP stimulation appears to depend on the -45 region sequence as our -45 region mutant s demonstrate different responses to HL159 CAP stimulation in vivo. (C ) 1998 Academic Press.