We will use biochemical and genetic approaches to analyze the mechanism of promoter utilization by the three DNA-dependent RNA polymerases required during bacteriophage N4 development. Early N4 RNAs are transcribed by a virion-encapsulated, phage- coded, rifampicin-resistant RNA polymerase (vRNAP) which recognizes its promoters on single-stranded DNA with in vivo specificity. Promoter recognition in vivo requires supercoiled template and E. coli single-stranded DNA binding protein (SSB). The role of supercoiling and SSB in activation of N4 vRNAP promoters will be determined by footprinting, affinity chromatography and the isolation of SSB mutants specifically altered in N4 early RNA synthesis. The gene for the vRNAP will be cloned in order to eventually manipulate it to determine functional domains in the protein. The fate of the vRNAP after injection will be determined. Middle N4 RNAs are synthesized by a second phage-coded, rifampicin- resistant RNA polymerase (N4 RNAP II) composed of two polypeptides 30,000 and 40,000 MW which, in vitro, does not utilize double- stranded DNA. A third polypeptide, 15,000 MW, is required in vivo. Recently, we have determined the in vivo sites of transcription initiation and developed an in vitro system which utilizes these sites in vitro and is dependent on the activity of the 15,000, 30,000 and 40,000 MW polypeptides. We will determine the relevance of the conserved sequences at the sites of transcription initiation to promoter activity by deletion and mutation analysis. The 15,000 MW polypeptide will be purified, and its role in N4 RNAP II promoter recognition studied. The N4 RNAP II polypeptides will be cloned in order to initiate structural studies. Late N4 transcription requires the activity of the E. coli RNA polymerase. Sites of late transcription initiation do not show homology at -35 and -10, a conserved octanucleotide is located just upstream of +1. These sites are poor templates for E. coli RNA polymerase when present on linear DNA. They are activated upon template supercoiling. Recent results suggest that the N4-coded single-stranded DNA binding protein (DBP) is required. The kinetic parameters of N4 late promoters will be determined to gain insights into the role of supercoiling in promoter activation. The interaction of the N4 DBP with late promoters will be studied. We will attempt to isolate mutants in the N4 DBP which specifically affect N4 late transcription.
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