Rj. Noel et Ws. Reznikoff, CAP, THE -45 REGION, AND RNA-POLYMERASE - 3 PARTNERS IN TRANSCRIPTIONINITIATION AT LACP1 IN ESCHERICHIA-COLI, Journal of Molecular Biology, 282(3), 1998, pp. 495-504
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.