Intersubunit interaction changes resulting from cAMP activation of CRP

Dimeric cyclic adenosine 3',5' monophosphate receptor protein (CRP) complexes with cAMP to form an asymmetric CRP:(cAMP)1 complex. CRP:(cAMP)1 binds upstream of lacP to activate the promoter via interactions with RNA polymerase. The goal of this work was to determine whether the cAMP-bound subunit of CRP:(c AMP)I must be positioned exclusively in one orientation with respect to RNA polymerase to activate transcription. In vitro transcription experiments were designed to determine whether the cAMP-bound subunit must be oriented in either the promoter-proximal or the promoter-distal position to activate transcription.

Site-directed mutagenesis was used to construct a CRP species that had altered affinity for DNA binding and decreased affinity for cAMP. Incubation of this mutant with WT CRP produced a heterodimeric species containing one WT subunit and one mutant subunit. At low concentrations of cAMP, only the WT subunit binds cAMP, producing CRP:(cAMP)i. The differing subunit affinities for specific DNA sequences allowed for orientation of CRP:(cAMP)i on two asymmetric templates. Mutagenesis of the CRP consensus binding sequence produced, via point mutations in either CRPbinding half-site, two templates capable of orienting the cAMP-bound subunit in either the promoter-proximal or promoter-distal position. In vitro transcription assays showed that the heterodimer activated transcription with the cAMP-bound subunit in either orientation. This data indicates the CRP:(cAMP)1 complex is not required to orient its cAMP-bound subunit with respect to RNA polymerase to activate lacP.