Upstream repression and CRP stimulation of the Escherichia colil-arabinose operon

Repression of the Escherichia coli araBAD promoter, P_BAD, was studied using a mutant p_BAD promoter (cip-5) that is expressed in the absence of the two proteins required for P_BAD induction, AraC protein and the cyclic AMP receptor protein (CRP-cAMP). Like the wild type promoter, cip-5 was repressed by AraC protein, and this repression required a site well upstream of the transcriptional start site. cip-5 was used to determine whether repression results from interference with the functioning of either AraC protein at araI and/or CRP-cAMP. Repression of cip-5 was eliminated by a point mutation within the AraC protein binding site araI but was not affected in the absence of CRP-cAMP. These results suggest that repression involves an interaction between two AraC protein binding sites located over 200 nucleotides apart. Our results also suggest that the majority of the CRP requirement for P_BAD is a result of P_BAD repression. When repression was abolished by deletion of the araO₂ site, the requirement for CRP-cAMP in P_BAD induction was greatly reduced.