The proposed research involves study of nucleic acid-protein interactions through application of physical biochemical techniques to systems of central interest in molecular biology. Much of the research will deal with components of the E. coli lactose operon regulatory mechanism. Studies will be made of the binding of RNA polymerase and of cyclic AMP binding protein (CAP) to various DNAs, to determine binding site size, base sequence specificity, single- or double-strand preference, and association constants under various solvent conditions. The methods will be those which have been used to study non-specific binding of lac repressor to DNA. These include thermal melting, sedimentation, and optical methods (circular dichroism, ultraviolet spectroscopy, etc.) The same approaches will then be applied to interactions in the RNA polymerase-CAP-DNA system, with the aim of elucidating details of the molecular mechanism whereby CAP stimulates RNA polymerase, and hence transcription, at catabolite sensitive operons. The results will shed light on the role of non-specific binding as a modulator of CAP and RNA polymerase function (non-specific binding of lac repressor recently has been shown to occur in vivo). It is planned to eventually extend these studies to interactions of the lac regulatory proteins with their specific functional sites on DNA (which should be obtainable by recombinant DNA techniques), A similar approach will be used to investigate the assembly of bacteriophage P22, which infects S. typhimurium. Physical techniques (primarily, but not exclusively) will be used to study association of the major head proteins with DNA. The results should be of interest with respect to the molecular mechanisms and forces involved in assembling and stabilizing viruses. A long term reason for studying phage assembly is that this process is unique to the virus and hence may be attacked by drug therapy with minimal damage to the host cell. These measurements may also give results of general application to protein-DNA interactions and thus will complement our studies on regulatory proteins.
NIH ExPorter
NIH ExPorter