The long-term objective of this research is an understanding at the molecular level of the equilibria and kinetics of the interactions of E. coli RNA polymerase and DNA that are involved in the control of gene expression. In a broader sense, we are interested in understanding the contributions of ionic, hydrophobic and other noncovalent interactions to the thermodynamcs and mechanisms of the binding of proteins and model cationic ligands to nucleic acids. Our work to date has focused on the measurement and interpretation of binding constants for the interactions of E. coli RNA polymerase, lac repressor, and model ligands with DNA sites. We have gained a quantitative understanding of the roles of ion release and ionic interactions in stabilizing the specific and nonspecific complexes of polymerase and repressor with DNA. Here I propose further experimental and interpretative work on the equilibria and kinetics of the interaction of RNA polymerase and DNA. Double label nitrocellulose filter binding experiments will be used to measure binding constants and rate constants for the specific interactions of RNA polymerase with promoter-containing restriction fragments of DNA (the T7 DNA A1 promoter; the lac promoter) as a function of temperature, pH, and ionic conditions. In addition, filter binding and sedimentation studies of the nonspecific binding of core and holoenzyme forms of RNA polymerase, and sedimentation velocity studies on RNA polymerase aggregation, are proposed.
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