The studies proposed in this application are aimed at understanding the mechanisms for late gene expression in the temperate coliphage P2. P2 late promoters do not resemble sequences normally recognized by E. coli RNA polymerase, although the host enzyme is required for late transcription. Normal expression of P2 late genes requires the P2 DNA replication genes and the product of the P2 ogr gene. Transactivation of P2 late genes by P4 requires the product of the P4 delta gene and initiates transcription at the same sites as normal P2 late gene expression. The functional domains of P2 late gene promoters will be defined by deletion analysis and in vitro mutagenesis. The interactions between purified P2 ogr protein, late promoters and host RNA polymerase will be investigated using chemical and kinetic methods. An additional P2-encoded factor which appears to be required for expression of P2 late genes will be isolated and its effect on late gene transcription will be studied in vitro. A point mutation in the alpha subunit of E. coli RNA polymerase specifically blocks P2 late gene transcription. Additional RNA polymerase mutants which map to the alpha subunit and affect P2 late transcription will be isolated and characterized. The effects of amino acid substitution in the ogr protein will be studied using different amber suppressors. The interactions between temperate coliphage P2 and satellite phage P4 provide a unique model system for studying the modulation of promoter specificity of E. coli RNA polymerase, and should lead to new insights into how RNA polymerase recognizes sequences in DNA.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM034651-06
Application #
3286007
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1985-09-05
Project End
1992-08-31
Budget Start
1990-09-01
Budget End
1992-08-31
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Virginia Commonwealth University
Department
Type
Schools of Medicine
DUNS #
City
Richmond
State
VA
Country
United States
Zip Code
23298
Christie, Gail E; Temple, Louise M; Bartlett, Becky A et al. (2002) Programmed translational frameshift in the bacteriophage P2 FETUD tail gene operon. J Bacteriol 184:6522-31
Linderoth, N A; Julien, B; Flick, K E et al. (1994) Molecular cloning and characterization of bacteriophage P2 genes R and S involved in tail completion. Virology 200:347-59
Ayers, D J; Sunshine, M G; Six, E W et al. (1994) Mutations affecting two adjacent amino acid residues in the alpha subunit of RNA polymerase block transcriptional activation by the bacteriophage P2 Ogr protein. J Bacteriol 176:7430-8
Ziermann, R; Bartlett, B; Calendar, R et al. (1994) Functions involved in bacteriophage P2-induced host cell lysis and identification of a new tail gene. J Bacteriol 176:4974-84
King, R A; Anders, D L; Christie, G E (1992) Site-directed mutagenesis of an amino acid residue in the bacteriophage P2 ogr protein implicated in interaction with Escherichia coli RNA polymerase. Mol Microbiol 6:3313-20
Birkeland, N K; Lindqvist, B H; Christie, G E (1991) Control of bacteriophage P2 gene expression: analysis of transcription of the ogr gene. J Bacteriol 173:6927-34
Temple, L M; Forsburg, S L; Calendar, R et al. (1991) Nucleotide sequence of the genes encoding the major tail sheath and tail tube proteins of bacteriophage P2. Virology 181:353-8
Linderoth, N A; Ziermann, R; Haggard-Ljungquist, E et al. (1991) Nucleotide sequence of the DNA packaging and capsid synthesis genes of bacteriophage P2. Nucleic Acids Res 19:7207-14
Grambow, N J; Birkeland, N K; Anders, D L et al. (1990) Deletion analysis of a bacteriophage P2 late promoter. Gene 95:9-15
Christie, G E; Calendar, R (1990) Interactions between satellite bacteriophage P4 and its helpers. Annu Rev Genet 24:465-90

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