The overall object of the proposed research is (a) to identify, purify and extensively characterize the Escherichia coli proteins that are necessary for the lytic growth of bacteriophage Lambda, (b) to identify and purify the phage gene products that interact with them, and (c) to elucidate the exact mechanism of the interactions between the host and phage proteins. The various E. coli functions, originally identified through the isolation of mutants that block the ability of to propagate, have been shown to fall into three major groups: those required for Lambda DNA replication, Lambda RNA transcription and phage morphogenesis. A combination of genetic and biochemical analysis will be employed in order to elucidate the exact mode of action of all these functions. The genetic experiments will include (a) the isolation and characterization of mutations with discernible phenotypes (Cs, Ts or Nonsense) in the genes already identified as well as in any remaining to be discovered, and (b) the isolation and characterization of extragenic suppressors among these various genes. The biochemical approach will include (a) the cloning, overproduction and purification of wild type as well as mutant bacterial and phage gene products, (b) the demonstration of potential protein-protein interactions among the purified gene products using a variety of approaches that include cosedimentation, cross-linking and affinity chromatography, and (c) the design of in vitro reconstituted systems for DNA replication, RNA transcription and morphogenesis that are capable of specifically detecting the activities of these gene products.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM023917-09
Application #
3271947
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1977-04-01
Project End
1990-03-31
Budget Start
1985-04-01
Budget End
1986-03-31
Support Year
9
Fiscal Year
1985
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
Zeilstra-Ryalls, J; Fayet, O; Georgopoulos, C (1996) In vivo protein folding: suppressor analysis of mutations in the groES cochaperone gene of Escherichia coli. FASEB J 10:148-52
Wall, D; Zylicz, M; Georgopoulos, C (1995) The conserved G/F motif of the DnaJ chaperone is necessary for the activation of the substrate binding properties of the DnaK chaperone. J Biol Chem 270:2139-44
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
Osipiuk, J; Georgopoulos, C; Zylicz, M (1993) Initiation of lambda DNA replication. The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein. J Biol Chem 268:4821-7
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
Ziegelhoffer, T; Yau, P; Chandrasekhar, G N et al. (1992) The purification and properties of the scaffolding protein of bacteriophage lambda. J Biol Chem 267:455-61
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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