D. Czarniecki et al., THE -45-REGION OF THE ESCHERICHIA-COLI LAC PROMOTER - CAP-DEPENDENT AND CAP-INDEPENDENT TRANSCRIPTION, Journal of bacteriology, 179(2), 1997, pp. 423-429
The lactose (lac) operon promoter is positively regulated by the catab
olite gene activator-cyclic AMP complex (CAP) that binds to the DNA lo
cated 61.5 bp upstream of the transcription start site. Between the CA
P binding site and the core promoter sequence is a 13-bp sequence (fro
m -38 to -50 [the -45 region]). The possible roles of the -45 region i
n determining the CAP-independent level of lac expression and in the C
AP activation process were studied by isolating and characterizing ran
dom multisite mutations. Only a small percentage of mutants have drama
tic effects on lac promoter activity. Among the mutations that did aff
ect expression, a 26-fold range in lac promoter activity in vivo was o
bserved in the CAP-independent activity. The highest level of CAP-inde
pendent lac expression (13-fold the level of the wild-type lac promote
r) correlated with changes in the -40 to -45 sequence and required an
intact RNA polymerase cr subunit for in vitro expression, as expected
for an upstream DNA recognition element. Mutant promoters varied in th
eir ability to be stimulated by CAP in vivo, with levels ranging from
2-fold to the wild-type level of 22-fold. Only a change of twofold in
responsiveness to CAP could be attributed to direct DNA sequence effec
ts. The -40 to -45 sequence-dependent enhancement of promoter activity
and CAP stimulation of promoter activity did not act additively. The
mutant promoters also displayed other characteristics, such as the act
ivation of nascent promoter-like activities overlapping Inc P1 and, in
one case, replicon-dependent changes in promoter activity.