The proposed projects will use a combination of genetics, biochemistry, and structural analysis to study protein-protein and protein-nucleic acid interactions and their roles in regulating gene expression. We will study two systems: the SOS regulatory system, and regulation of coliphage hk022 gene expression. The SOS regulatory system is controlled by the interplay of two proteins, the LexA repressor and the RecA protein. When the regulatory system is induced by DNA damage, RecA is activated to a form which interacts with LexA repressor, leading to proteolytic cleavage of LexA. However, this cleavage is unusual in that RecA appears to stimulate repressor self-cleavage, rather than itself acting as a protease. It is proposed to explore the interaction between the two proteins in detail. Mutations in both genes will be isolated which affect the interaction between the two proteins. Methods will be developed to measure the strength of the interaction, both in the wild-type case and with mutant proteins. The other interesting function of LexA is its specific DNA binding. This appears to be unusual, in that LexA appears able to dimerize on the DNA under some conditions, a feature which will be explored in detail. The properties of mutant proteins with altered DNA-binding specificity will also be studied. Finally, it is planned to isolate high-quality crystals of LexA and complexes with the operator and with activated RecA for use in x-ray crystallographic analysis. Structural information gained from this analysis can be correlated with the genetic and biochemical data. The second regulatory system to be studied, gene control in coliphage hk022, is of interest for two reasons. First, the cI repressor of this phage binds to adjacent operators with a very high degree of cooperativity, far higher than that of phage lambda. It is planned to analyze this cooperative binding in detail. Mutants affecting cooperativity will be isolated. Chimeric lambda-hk022 cI proteins will be isolated and studied. Second, the molecular mechanisms controlling the lysis-lysogeny decision in hk022 appear to differ from those of phage lambda. The roles of hk022 cI and Cro proteins in this regulatory decision will be explored in detail. These studies are of wide general interest for the insights they offer into mechanisms of gene regulation, and to our understanding of protein-protein and protein-DNA interactions, cooperative DNA binding, and the question of how a protein-protein interaction can be transduced into stimulating a catalytic reaction.