Genetic and Biochemical Analysis of Specific LexA Cleavage

该项目的总体目标是大肠杆菌LexA蛋白的结构和功能分析。这种蛋白质经历一种称为自体消化的自裂反应，使其抑制功能失活。在体内，卵裂需要另一种蛋白质RecA的激活形式。体内数据表明，RecA不是作为一种经典的蛋白酶，而是通过刺激LexA自身消化而起作用。本研究将采用遗传学、生物化学和蛋白质化学的方法，进一步分析自体消化的机制和RecA在刺激这一反应中的作用。将分离lexA和recA的突变，这些突变要么影响两种蛋白质之间的相互作用，要么影响一个或两个切割反应的速率。特别是，将研究比野生型LexA切割更快的突变蛋白，目的是确定和分析切割中的限速步骤。计划用操作器和活化的RecA分离LexA晶体和配合物，用于x射线晶体学分析。从这种分析中获得的结构信息可以与遗传和生化数据相关联。这些研究具有普遍意义，因为它们提供了对自我加工反应的见解，这些反应在广泛的重要生物分子中被观察到，包括自剪接RNA、生长因子受体、病毒多蛋白和癌基因产物。

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.