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.
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