Genetic and Biochemical Analysis of Specific LexA Cleavage

该项目的总体目标是对大肠杆菌的LexA蛋白进行结构和功能分析。这种蛋白质经历了一种被称为自体消化的自我切割反应，使其抑制物功能失活。在体内，卵裂需要另一种蛋白质RecA的激活形式。在VITO中的数据表明，RecA的作用不是作为一种经典的蛋白酶，而是通过刺激LexA的自我消化。这项拟议的研究将结合遗传学、生物化学和蛋白质化学，进一步分析自动消化的机制以及RecA在刺激这一反应中的作用。LexA和recA的突变将被分离出来，这些突变会影响两种蛋白质之间的相互作用，或者影响一种或两种切割反应的速度。特别是，将研究比野生型LexA更快切割的突变蛋白，目的是识别和分析切割中的限速步骤(S)。它计划分离LexA的晶体以及与操作符和活化的RecA的络合物，用于X射线结晶学分析。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。这些研究提供了对自我处理反应的洞察，这些研究引起了普遍的兴趣，这些反应在广泛的重要生物分子中观察到，包括自剪接RNA、生长因子受体、病毒多蛋白和癌基因产品。

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.