A genetic approach will be used to analyze the specific chemical contacts made between the E. coli Trp repressor and its 20 base pair operator, and the bacteriophage Gamma repressor and its 17 base pair operator. Oligonucleotide-directed localized random mutagenesis will be used to alter each of the condons in the two repressor genes representing amino acid residues thought to be critical for DNA binding. In this way, pools of mutant plasmid producers of each repressor will be constructed. Each pool will include plasmids that produce repressors differing at a single amino acid residue position and that carry each of the 20 possible amino acids at this position. From each pool, we will select mutant repressors that have novel DNA binding properties. altered specificity and hyperrepressor mutants will be selected by virtue of their ability to repress operators that are insensitive to the binding of wild-type repressor, and distinguished by their ability or inability to bind the wild-type operator. In addition, we will select or screen dominant negative mutations in each repressor gene that specifically interfere with binding to the wild-type operator. The ability of each mutant repressor to bind a complete set of mutant operators carrying each possible pair of symmetric base pair substitution will be tested. Mutations that confer informative DNA binding phenotypes will be defined precisely by DNA sequence analysis. In collaboration with other laboratories, we will determine the DNA binding properties of the informative mutant repressors in vitro. The long-term goal of this research is an understanding of the chemical basis of gene expression-- how proteins recognize specific nucleic acid sequences. The understanding of gene regulation is central to the understanding of normal development in higher organisms and of the diseases that result when these regulatory processes go awry.
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