Genetic and Biochemical Analysis of Specific LexA Cleavage

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The overall goal of the project is a structural and functional analysis of the LexA protein of Escherichia coli. This protein undergoes a self-cleavage reaction, termed autodigestion, which inactivates its repressor function. In vivo, cleavage requires an activated form of another protein, RecA. In vito data indicate that RecA acts, not as a classical protease, but by stimulating LexA autodigestion. The proposed research will analyze further the mechanism of autodigestion and the role of RecA in stimulating this reaction, using a combination of genetics, biochemistry, and protein chemistry. Mutations in lexA and recA will be isolated which affect either the interaction between the two proteins, or the rates of one or both cleavage reactions. In particular, mutant proteins which cleave faster than wild-type LexA will be studied, with the goal of identifying and analyzing the rate-limiting step(s) in cleavage. It is planned to isolate crystals of LexA and of 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. These studies are of general interest for the insights they offer into self-processing reactions, which are observed in a wide range of important biological molecules, including self- splicing RNA, growth factor receptors, viral polyproteins, and oncogene products.