BACTERIAL FUNCTIONS IN PHAGE DEVELOPMENT

这个提议的总体目标是从分子的细节上理解 宿主参与λ噬菌体裂解生长的机制。 重点是了解大会的进程， 分解复杂的蛋白质结构，导致λ DNA 复制，或λ头的形态发生。 的一些具体 目的包括（a）确定dnaK、dnaJ和 grpE蛋白分解在ori λ处组装的蛋白-DNA复合物， 从而允许DNA复制的起始。 这包括确切的 在dnaK、dnaJ和grpE存在下， (b)β-daO蛋白水解在允许双向 复制的 这些研究包括纯化ATP依赖的 （c）蛋白酶的可能作用， 腺苷二磷酸磷酸化和潜在的ATP结合在调节细胞凋亡中的作用 （d）确定和说明其他 E.大肠杆菌基因，其产物在λ DNA中起直接作用， 复制，在功能上与dnaK，dnaJ相互作用或取代dnaK，dnaJ 或grpE蛋白质，（e）完成对所述重组蛋白的纯化， 形态发生蛋白，（f）试图组装β-dodecameric， 在由groES构成的限定系统中的头-尾连接器结构， groEL、p53 daNu 3和p53 daB纯化蛋白，（g）测试我们的“齿轮” groEs和groEL蛋白质-蛋白质相互作用机制的模型。 这将包括前内酰胺酶的体外折叠研究 酶，（h）纯化并检查突变体的生化性质 groES和groEL多肽的结构/功能分析，以及（i）所述多肽的结构/功能分析。 groES和groEL蛋白。 这将主要通过排序来完成 groES-和groEL-突变，包括对已经存在的 鉴定了groES* 和groEL* 补偿突变。

The overall goals of this proposal are to understand in molecular detail the mechanisms of host participation n the lytic growth of phage lambda. Emphasis is placed on understanding the process of the assembly and disassembly of complex protein structures, leading either to lambda DNA replication, or the morphogenesis of the lambda head. Some of the specific aims include (a) determination of the mechanism by which the dnaK, dnaJ and grpE proteins disassemble the protein-DNA complex assembled at ori lambda, thus allowing the initiation of DNA replication. This includes the exact mechanism of lambdaP sequestration in the presence of dnaK, dnaJ and grpE, (b) the role that lambdaO proteolysis plays in allowing bidirectional replication. These studies include purification of the ATP-dependent protease(s) responsible for lambdaO cleavage, (c) the possible role of lambdaO phosphorylation and potential ATP binding in modulating the replication process, (d) identification and characterization of additional E. coli genes whose products play either a direct role in lambda DNA replication, functionally interact with, or substitute for the dnaK, dnaJ or grpE proteins, (e) finishing the purification of the lambdaB morphogenetic protein, (f) attempting to assemble the lambdaB dodecameric, head-tail connector structure in a defined system consisting of groES, groEL, lambdaNu3 and lambdaB purified proteins, (g) testing our "cogwheel" model of the mechanism of groEs and groEL protein-protein interaction. This will include studies with the in vitro folding of the pre-lactamase enzyme, (h) purifying and examining the biochemical properties of mutant groES and groEL polypeptides, and (i) structure/function analysis of the groES and groEL proteins. This will be done chiefly through the sequencing of groES- and groEL- mutations, including an analysis of the already identified groES* and groEL* compensatory mutations.