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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM023917-15
Application #
3484498
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-04-01
Project End
1995-03-31
Budget Start
1991-04-01
Budget End
1992-03-31
Support Year
15
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of Utah
Department
Type
Schools of Medicine
DUNS #
City
Salt Lake City
State
UT
Country
United States
Zip Code
84112
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Zeilstra-Ryalls, J; Fayet, O; Georgopoulos, C (1994) Two classes of extragenic suppressor mutations identify functionally distinct regions of the GroEL chaperone of Escherichia coli. J Bacteriol 176:6558-65
Wall, D; Zylicz, M; Georgopoulos, C (1994) The NH2-terminal 108 amino acids of the Escherichia coli DnaJ protein stimulate the ATPase activity of DnaK and are sufficient for lambda replication. J Biol Chem 269:5446-51
Zeilstra-Ryalls, J; Fayet, O; Baird, L et al. (1993) Sequence analysis and phenotypic characterization of groEL mutations that block lambda and T4 bacteriophage growth. J Bacteriol 175:1134-43
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Ziemienowicz, A; Skowyra, D; Zeilstra-Ryalls, J et al. (1993) Both the Escherichia coli chaperone systems, GroEL/GroES and DnaK/DnaJ/GrpE, can reactivate heat-treated RNA polymerase. Different mechanisms for the same activity. J Biol Chem 268:25425-31
Liberek, K; Galitski, T P; Zylicz, M et al. (1992) The DnaK chaperone modulates the heat shock response of Escherichia coli by binding to the sigma 32 transcription factor. Proc Natl Acad Sci U S A 89:3516-20
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Liberek, K; Skowyra, D; Zylicz, M et al. (1991) The Escherichia coli DnaK chaperone, the 70-kDa heat shock protein eukaryotic equivalent, changes conformation upon ATP hydrolysis, thus triggering its dissociation from a bound target protein. J Biol Chem 266:14491-6

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