On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions

Abstract

Using DNase footprinting and transcription assays in vitro we have probed the effect of the cAMP-cAMP receptor protein complex (cAMP-CRP) on the positioning of RNA polymerase and on the location of the transcription start point at the Escherichia coli gal and lac operon regulatory regions. In both cases, RNA polymerase can form two alternative complexes which promote transcription from two different start points, S1 and S2: pre-incubation of promoter DNA with cAMP-CRP results in a shift of the transcription start from S2 to S1 and in an increase in the rate of open complex formation. Moreover, the rate of formation of each heparin-resistant complex parallels the establishment of the corresponding footprint, showing that the stable binding corresponds to open complex formation. We show that, in the case of gal, RNA polymerase, which is bound so as to transcribe from S2, cannot be diverted to S1 by subsequent addition of cAMP-CRP. In contrast, in the case of lac, when cAMP-CRP is added after RNA polymerase, complexes which initiate transcription at S2 are rapidly converted to complexes which initiate at S1. Finally, we present data which suggest that protein-protein interactions are essential for CRP-induced activation at both the lac and gal promoters.