Genetic and Biochemical Analysis of Specific LexA Cleavage

9305092用破坏DNA或抑制复制的试剂对大肠杆菌进行少量处理会触发SOS反应，这是对损害的全球性反应，通过各种手段促进细胞存活。这种反应涉及两种调节蛋白的作用：LexA和RecA，前者在正常生长过程中抑制约20个SOS基因，后者通过诱导处理激活LexA抑制物，使其失活并诱导SOS基因的诱导。因此，特定的切割在SOS反应中起着至关重要的作用。除了LexA裂解的这种中心调节作用外，这种反应还具有相当大的生物化学兴趣。它的机制是不寻常的，因为LeA在一种称为自身消化的分子内反应中自我裂解，并在生理条件下激活RecA来刺激LexA的自我裂解。在这些条件下，LexA裂解被设计为缓慢进行，但同时能够大幅增加速率。这一观点提出了两个相关的问题：LexA自动消化的机制是什么，RecA蛋白如何促进这一反应？为解决这些问题，提出了三种补充办法。第一个是继续分离和鉴定影响LexA裂解速度的突变。第二种方法是继续发展双分子LexA裂解反应，在该反应中，LexA或lambda抑制子的一个分子作为酶来裂解LexA的其他分子。第三种方法是寻找物理证据，证明LexA可以以两种不同的构象状态存在。越来越多的生物重要反应正在被发现，这些反应都是分子内的。其中包括自剪接RNA内含子、趋化蛋白的自磷酸化和自磷酸酶活性、癌基因蛋白、生长因子受体和其他重要蛋白的自磷酸化。这项工作对于分子内自处理反应的性质，以及通过与其他效应分子的相互作用来调节这些反应的额定值的方法，具有普遍意义。***

9305092 Little Treatment of E. coli by agents that damage DNA or inhibit replication triggers the SOS response, a global response to the damage that promotes cell survival by a variety of means. This response involves the action of two regulatory proteins: LexA, which represses about 20 SOS genes during normal growth, and RecA, which is activated by inducing treatments to a form that catalyzes the specific cleavage of LexA repressor, inactivating it and leading to induction of the SOS genes. Hence, specific cleavage plays a crucial role in the SOS response. In addition to this central regulatory role of LexA cleavage, this reaction is of considerable biochemical interest. Its mechanism is unusual, in that LeA cleaves itself in an intramolecular reaction termed autodigestion, and activated RecA acts to stimulate LexA self-cleavage under physiological conditions. In these conditions, LexA cleavage is designed to proceed slowly, but at the same time to be capable of large rate increases. This view raises two related questions: What is the mechanism of LexA autodigestion, and how can RecA protein facilitate this reaction? Three complementary approaches are proposed to address these questions. The first is to continue to isolate and characterize mutations that influence the rate of LexA cleavage. The second approach is to continue to develop the bimolecular LexA cleavage reaction, in which one molecule of LexA or lambda repressor acts as an enzyme to cleave other molecules of LexA. The third approach is to look for physical evidence that LexA can exist in two different conformational states. %%% An increasingly large number of biologically important reactions are being discovered that are intramolecular. These include self- splicing RNA introns, autophosphorylation and autophosphatase activity of chemotaxis proteins, and autophosphorylation of oncogene proteins, growth factor receptors, and other important proteins. The proposed work is of general signifi cance for the insights it is expected to yield regarding the properties of intramolecular self-processing reactions, and the means by which the rated of these reactions can be regulated by interactions with other effector molecules. ***