BACTERIAL FUNCTIONS IN PHAGE DEVELOPMENT

这个提议的总体目标是在分子细节上理解 噬菌体Lambda裂解生长的寄主参与机制。 重点是了解组装过程和 复杂蛋白质结构的分解，导致形成lambda DNA 复制，或板头的形态发生。一些具体的 目标包括：(A)确定dNAK、dNAJ和 GRPE蛋白质分解在Ori lambda组装的蛋白质-DNA复合体， 从而允许DNA复制的启动。这包括准确的 DNAK、DNAJ和GRPE存在下LambdaP的固定机理 (B)lambdaO蛋白分解在允许双向的过程中所起的作用 复制。这些研究包括提纯依赖于ATP的 负责LambdaO裂解的蛋白酶(S)，(C)可能的作用 LambdaO磷酸化和潜在的ATP结合在调节细胞周期中的作用 复制过程，(D)确定和表征其他 其产物直接作用于lambda DNA的大肠杆菌基因 复制、在功能上与dna K、dna J交互或替代dna 或GRPE蛋白，(E)完成lambdaB的纯化 形态发生蛋白，(F)试图组装lambdaB十二聚体， 在由凹槽组成的限定系统中的头尾连接器结构， GroEL、lambdaNu3和lambdaB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。 这将包括对前内酰胺酶体外折叠的研究。 酶，(H)纯化和检测突变体的生化特性 GroES和GroEL多肽，以及(I)结构/功能分析 Groes和GroEL蛋白。这将主要通过测序来完成 Groes-和GroEL-突变，包括对已经 确定了Groes*和GroEL*代偿性突变。

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.