BACTERIAL FUNCTIONS IN PHAGE DEVELOPMENT

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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.