Quantitative Studies of the Biotin Regulatory System

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DESCRIPTION (provided by applicant): Protein-protein interactions are critical to regulation of expression of genetic information. The strength of homologous protein-protein interactions dictates the level of occupancy of transcriptional regulatory sites on DNA. Patterns of heterologous protein-protein interactions determine the selection of DNA target site and/or the level of occupancy of a site. Through their influence on DNA binding these macromolecular interactions are fundamental to control of expression of genetic information. Elucidation of the details necessary for successful clinical intervention at the level of protein-protein interactions in control of gene expression requires combined solution physico-chemical studies of function as well as high-resolution structural information. The Escherichia coli biotin regulatory system provides an excellent model system for detailed studies of the control of a genetic regulatory switch via protein-protein interactions. In this proposal experiments designed to determine the structural and thermodynamic features of corepressor-induced assembly of a transcriptional repressor are described. Strategies to examine the control of gene expression via heterologous protein-protein interactions are also outlined. Methods utilized in the proposed work include site-directed and random mutagenesis, stopped-flow fluorescence kinetic measurements, sedimentation equilibrium, DNaseI footprinting, isothermal titration calorimetry and x-ray crystallography. Results of these combined studies will yield information of general significance to understanding the detailed chemistry of the protein-protein interactions essential to regulation of genetic expression.