E.coli cyclic AMP receptor protein (CRP) is the key regulatory factor responsible for the bacteria to adapt to a change in the source of energy. CRP controls the expression of more than 20 genes which code the proteins that metabolize the sugars for energy. This laboratory has developed a research program to quantitatively link the various reactions that are involved in the regulatory activity of CRP by monitoring many of the individual reactions. These reactions include the activation of CRP by cAMP binding, the structural changes in CRP induced by cAMP, the binding of CRP-cAMP complex to the specific DNA site, the induced DNA bending and the binding of RNA polymerase to the DNA-CRP-cAMP complex. Results of this study indicate that the asymmetric nature of the CRP-cAMP complex may be an important feature in recognizing the specific DNA binding site in different operons. It is proposed that the asymmetric CRP-cAMP complex will utilize its asymmetric nature to orient itself with the non-palindromic DNA half sites. Since the DNA binding sites in different operons assume different orientations, the binding of an asymmetric CRP-cAMP to the DNA binding site can potentially lead to a variety of different surfaces on CRP exposed for interaction with RNA polymerase or other transcription factors. This might be the mechanism for CRP to distinguish the more than 20 genes regulated by CRP. Using a combination of hybrids of CRP dimer specifically engineered to be asymmetric in cAMP and DNA binding, the specific orientation adopted by CRP in the DNA-protein complex will be identified. Furthermore, the roles of the sequence difference in the DNA half-sites will be probed by measuring the protein-DNA binding constant and dissociation rate constant in addition to the affinity for the formation of the DNA-CRP-RNA polymerase complex. The ability of the DNA half-sites to induce conformational changes in CRP will also be monitored. Results of this investigation should provide direct evidence to assess the role of asymmetry in the regulation of gene expression of CRP. Since the formation of the asymmetric CRP-cAMP complex is the central issue, it is proposed to elucidate the mechanism of activation of CRP with the help of mutants generated by site-directed mutagenesis. The sites of mutation have been identified by natural selections and these mutants are products of single site mutations with different phenotypic characteristics. These sites are located at different parts of CRP. Thus, they might interfere with different parts of the pathway of CRP activation induced by cAMP. Tracking the structural perturbation with functional assays could provide significant insight to the mechanism of activation.
