Regulation of the expression of genetic information is critical to the proper development and functioning of any organsim. Regulation at the level of transcription initiation has been demonstrated to depend on macromolecular interactions formed between multiple proteins and target sites on the DNA. A mechanistic understanding of transcriptional regulation therefore requires detailed quantiative information about the individual macromolecular interactions that contribute to the process. The biotin regulatory system from Escherichia coli is an example of a complex transcriptional regulatory system in which physiological function depends on a combination of protein-small molecule, homologous and heterologous protein-protein and protein-DNA interactions. Thermodynamic coupling moreover, exists between sthe individual interactions. The goals of this reserch are to elucidate the structural, thedrmodynamic and kinetic rules that govern the physiological function in the biotin transcriptional regulatory system. The kinetics of cooperative binding of the major regulatory protein, the bioyin repressor, to its target site on the DNA, the biotin operator, will be measured using Time Resolved Dnase Footprinting and Stopped-Flow Fluorescence. Direct Equilbrium Fluorescence Titrations and Equilibrium Analytical Ultracentrigugation Measurements will be utilized to examine coupling of DNA binding to protein dimerization in assembly of the transcriptional repression complex. The structural elements of the protein and DNA involved in the multiple macromolecular interfaces formed in the transcriptional repression complex will be probed using Chemical DNA Footprinting and Chemical Protein Footprinting techniques. Single site mutants that disrupt one or more of these interfaces will be isolated and characterized o quantitate the consequences of altrations in the structural integrity of each interface for the functional energetics of the system. Finally, crystallization of complexes of the biotin repressor bound to small ligands and to DAN is proposed so that we may ultimately obtain high resolution structures of the multiple functional states of this allosteric site-specific DNA binding protein. Results of these proposed studies of the biotin regulatory system will provide information that is relevant to understanding the mechanism of function of any complex transcriptional regulatory system.
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