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. We are also addressing the issue of how many cAMP molecules must bind to CRP to activate it. We have made CRP heterodimers that contain one wild type subunit and one subunit that cannot bind cAMP. It appears that CRP can function with only one cAMP bound, although the subunit with the cAMP bound binds more easily to DNA. (ii) We are addressing how CRP activates transcription in vivo, since there appears to be far more CRP activation of lactose operon transcription in vivo than in vitro. We have isolated E. coli mutants that are defective in CRP activation of the lac operon; one of the mutations is in the gene encoding isocitrate dehydrogenase. A likely explanation for this is that accumulation of citrate or isocitrate prevents the lac operon from being expressed. We are currently searching for the target of the citrate/isocitrate toxicity. We have also found both in vitro and in vivo that the small DNA binding protein HU decreases the amount of lac transcription in the absence of CRP. Therefore, HU accounts for some of the discrepancy between and in vitro and in vivo results. - Escherichia coli, Gene regulation, Genetics, Transcription, Transcription Factors, metabolism, Metabolic Networks, Bacterial genetics, cyclic AMP, - Neither Human Subjects nor Human Tissues
