The objective of the proposed project is to achieve a detailed and precise understanding of the molecular mechanism of genetic regulation, at a level at which specific changes in the operation of this mechanism could be predicted and engineered. The ability to predict and control modifications to the function of genetic regulatory mechanisms will have far reaching consequences to the ability to treat genetic diseases and to use the mechanisms as targets for rational drug design, but existing knowledge is as yet insufficient to reach these goals. The trp-repressor from E. coli was chosen as a model system for detailed study, because it is one of the smallest and simplest complete regulatory systems available today. Starting with the known structures of the repressor protein, with and without the activator L-tryptophan, and of the ternary DNA-repressor-L-trp complex, it is possible to systematically investigate the role of each structural element (amino acid residue) in (1) maintaining specific contacts with the L-trp ligand and (2) with the operator DNA and (3) transmitting the signal regulating DNA binding between the binding sites. This will be achieved by studying point and multiple mutations which are known to alter the conformation and dynamics of the system. Very promising initial results have already been obtained with some of the mutants, providing clear directions for future studies. Correlation of the findings should allow an understanding of the signaling code and the design of the regulatory apparatus.
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