Dissecting the functional program of Escherichia coli promoters: the combined mode of action of Lac repressor and AraC activator

The mode of action of regulated promoters is largely determined by kinetic parameters which govern the interaction between promoters and proteins invo lved in induction and repression of transcription. To gain insight into the interplay between positively and negatively acting transcriptional regulat ors, in this case AraC and LacR, we have generated a panel of promoter sequ ences derived from Pl,c, the promoter of the Escherichia coli lac operon. T he function of these promoters is limited at different steps and to various extents within the pathway of RNA polymerase (RNAP)/promoter interaction. Moreover, in all promoters the cAMP receptor protein binding site was repla ced by the binding motif of AraC to prevent pleiotropic effects in vivo upo n activation. Analyzing the activation of these promoters by AraC in vivo u nder conditions of repression by LacR and derepression yielded a three step model of transcription initiation which reveals mechanisms of AraC and Lac R action. Our data show three distinct rate limiting steps at which AraC ca n exert its function. In general, the activator accelerates the formation o f the first stable complex between RNAP and promoter. At most promoter sequ ences, however, its main impact is on the conversion of the closed to the o pen complex. However, AraC is also capable of eliminating limitations at st eps following open complex formation.