DNA superhelical density and CRP-mediated lac promoter activation

The cyclic AMP receptor protein (CRP) functions in Escherichia coli in the regulation of several catabolite-sensitive operons. Cyclic AMP (cAMP) forms a complex with CRP which binds to specific DNA sites near the promoter of lactose operon {lacP) and activates the rate of its transcription by RNA polymerase. Mutant forms of the proteins (CRP*) that function independentiy of cAMP have been described. Studies were conducted to assess the response of the wild-type and mutant forms of CRP to changes in template DNA structure. To investigate the effect of varying template superhelical density (SHD) on CRP and CRP*-mediated lacP activity, in vitro transcription assays were performed. A lacP containing plasmid DNA, pJB3.5d/ac, was constructed and a series of topoisomers ranging from SHD = 0.000 to -0.098 were generated. The results of both full length transcripts and abortive initiation assays showed that CRP-mediated lacP activity was low at low template SHD and increased with increased template SHD under all conditions tested. Each mutant form of the protein was, however, unique in the range of DNA SHD providing maximal lacP stimulation. In addition, two mutant forms of CRP promoted RNA polymerase:/acP open complex formation in the absence of cAMP at half-times comparable to the CRPicAMP complex. The open complex formation half-times in reactions that contained wild-type CRPicAMP complex decreased with increasing SHD and are in good agreement with the published data. DNA:CRP binding properties of topoisomers were determined in experiments that utilize a product of in vivo recombination product, a small (451 base pairs) covalently closed circular DNA containing lacP region. The gel mobility shift assay on DNA:CRP:cAMP complex showed that in all DNA SHD examined, both wild-type and the mutant forms of CRP cause a retardation in migration rate, while in the absence of cAMP no difference in migration was observed. From the abortive initiation assay on 451 base pair (bp) DNA, it was found that lacP contained on the 451 bp circle does not show the same degree of cAMP dependence as lacP contained on a larger covalendy closed circle.