THE -45-REGION OF THE ESCHERICHIA-COLI LAC PROMOTER - CAP-DEPENDENT AND CAP-INDEPENDENT TRANSCRIPTION

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.