This research project is aimed at comprehending the mechanism by which genetic regulatory proteins function. THe lac and trp repressor proteins are representative of regulatory proteins in the classes of inducible and repressible systems in prokaryotes; both of these proteins can be isolated in the quantities required for physical and chemical studies. The specific objectives are 1) the determination of the effects of operator DNA on the core domain of the lac repressor protein, isolation of the core domain from mutant repressors, and determination of the effects of antiinducer on the binding properties of this protein fragment in order to further clarify the relative roles of the two separable regions of the lac repressor; 2) measurement of energy transfer between fluorophores within the structure of the protein and between probes attached to operator DNA and the protein in order to provide a crude 3-dimensional view of the lac repressor and its operator DNA interaction; 3) measurement of the thermodynamic parameters for denaturation and binding to ligands for the lac repressor by calorimetric methods; 4) determination of the binding parameters for trp repressor protein with its ligands over a wide range of conditions; 5) assessment of the effects of proteolytic enzymes on the trp repressor to determine whether this protein contains domains analogous to other DNA binding proteins; and 6) measurement of the intrinsic fluorescence properties of the trp repressor and this protein modified with fluorescent probes as a means of monitoring potential conformational changes upon binding to ligands and defining the environments surrounding the sites of these probes in the primary structure of the protein. The correlation of data obtained on these two systems may yield information regarding common characteristics of genetic regulatory proteins. Genetic regulation is an essential function in all living systems, and the currently known mechanisms involve protein-DNA interactions analogous to those operative in the trp and lac operons. Thus, the results from these studies not only form the base for understanding the mechanisms by which relatively simple systems operate but also for exploring similar functions in more complex systems.
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