DNA structure and cyclic AMP receptor protein-mediated lactose operon promoter activation



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Texas Tech University


The cyclic adenosine 3';5' monophosphate (cAMP) receptor protein (CRP) complexed with cAMP binds a site upsu-eam of the lactose operon promoter (lacP) and activates transcription of the operon. The binding of the wild-type and two mutant forms of CRP to lacF DNA has been characterized with respect to the CRP-induced DNA bending angle and the CRP bending locus.

DNase I footprint experiments were conducted to develop a better understanding of the requirements for the interaction of each of these three forms of CRP and lacP DNA. This study suggested that CRP-mediated transcription activation of lacP occurs through a mechanism that includes a role for protein-protein interaction between CRP and RNA polymerase.

An intramolecular DNA ligation assay was utilized to determine the DNA molar cyclization factor when bound by each of these three forms of CRP. Six DNA fragments, each containing the CRP binding site and a short phasing sequence, were constructed and served as substrates for this assay. Ligation assays were done in the absence of CRP, in the presence of either wild-type or mutant CRP, or in the presence of the wild-type or mutant CRP and cyclic nucleotide.



Genetic transcription, Operons, Deoxyribonucleic acid (DNA) polymerases, Cyclic adenylic acid, Promoters (Genetics)