QUANTITATIVE STUDIES OF THE BIOTIN REGULATORY SYSTEM

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Regulation of gene expression in prokaryotes and eukaryotes is effected by complex macromolecular assemblies. Function of these assemblies can depend on protein-protein, protein-small molecule, and protein-DNA interactions. Regulation of biotin biosynthesis and retention in E. coli depends on all three types of interactions. A model for the biotin system, based on a qualitative understanding of the macromolecular processes that contribute to the regulation, has been proposed. The biotin system provides an ideal model system for relating the physical chemistry of individual macromolecular interactions to the biology of a complex gene regulatory system. The goals of this research proposal are to: 1. perform quantitative studies of the interactions that contribute to biotin regulation to define the functional energetics of the biotin system. 2. investigate the thermodynamic driving forces responsible for the regulatory interactions. 3. evaluate and revise the qualitative model for biotin regulation in light of the results of the physical studies and develop and test quantitative models for the system. The techniques that will be employed in these studies include a. the quantitative DNAse footprint technique to measure DNA binding, b. equilibrium sedimentation to determine of subunit assembly properties, and c. fluorescence spectroscopy to measure heterologous protein-protein interactions. These studies of the biotin system will help define the relationship between the physical chemistry of individual biotin regulatory interactions and the biology of the entire system and may reveal general principles that are relevant to understanding more complex gene regulatory systems. Thermodynamic characterization of the interactions will, moreover, yield information about the physical chemical forces that drive these interactions.