The cyclic AMP (cAMP) receptor protein (CRP) is a DNA binding protein from Escherichia coli that can activate transcription when bound to specific sites located near promoters. CRP can serve as a model system for the study of gene regulation. We have been studying several steps in the pathway to CRP activation of transcription. (i) In order for CRP to become activated as a DNA binding protein, cAMP must bind to the protein and cause an allosteric change. We have isolated CRP mutants that are defective in the cAMP-induced allosteric change. In collaboration with Richard Brennan at the Oregon Health Sciences University, we are performing X-ray crystallographic analysis of one of the CRP mutants. To further assign residues responsible for the cyclic AMP-induced change in CRP, we have selected intragenic suppressor mutations for the allosteric defect. The positions of these suppressor mutations confirm our earlier hypothesis as to how the conformational change occurs in CRP. (ii) We have addressed the mechanism for transcription activation by CRP by three different approaches. A previous model for CRP activation of transcription stated that CRP activated by placing the alpha subunit of RNA polymerase down on the DNA in a particular manner. By constructing a promoter with both CRP binding sites and a binding site for the alpha subunit of RNA polymerase, we have shown that CRP activation of transcription occurs by a mechanism other than simple recruitment of RNA polymerase. To analyze transcription activation by CRP, we have developed a new method for determining the kinetic parameters of transcription initiation. Also, we have continued our studies on transcription activation on DNA that has a disruption between where CRP binds and the promoter. This disrupted DNA does not allow CRP activation of transcription because unwinding of the DNA at the promoter does not occur. We have also developed a protein footprinting assay that allows identification of regions within a protein involved in interacting with DNA or other proteins.
