Lrp binds to two regions in the dadAX promoter region of Escherichia coli to repress and activate transcription directly

The dadAX operon is expressed by multiple promoters that are repressed by l eucine-responsive regulatory protein (Lrp) and activated by cyclic AMP-CRP. In previous work, we found that alanine or leucine acted as inducers to an tagonize Lrp repression of the three major promoters directly. Here, we ide ntify 11 Lrp binding sites located within 350bp of dad DNA. A mutational an alysis, coupled with in vivo and in vitro transcription experiments, indica ted that Lrp sites that overlap the dad promoters were involved in repressi on. In contrast, sites upstream of the promoters did not appear to be neces sary for repression, but were required for activation by Lrp plus alanine o r leucine of one of the major dad promoters, P2. This activation by alanine or leucine was not simply relief of repression, as P2 transcription from a constitutive template was increased fivefold compared with the basal level of transcription found in the absence of Lrp and the co-activator cyclic A MP-CRP. Alanine or leucine decreased the affinity of Lrp to repressor sites , while having little or no effect on the binding of Lrp to activator sites . This differential effect of alanine and leucine on Lrp binding helps to e xplain how these modifiers influence both repression and activation of the dad operon.