We will use biochemical, genetic and physical approaches to analyze the structure, function, mechanism of promoter utilization and regulation of activity of two phage-coded, DNA-dependent RNA polymerases required during bacteriophage N4 development. Early N4 RNAs are transcribed by a virion-encapsulated, phage-coded, 320- kDa single-polypeptide RNA polymerase (vRNAP) which recognizes both a structure and specific sequences at its promoters on single-stranded template, i.e., it is a site-specific, single-stranded DNA binding protein. Promoter recognition on double-stranded DNA requires supercoiled template and E. coli single-stranded DNA binding protein (SSB). Activation is specific for E. coli SSB. The DNA structure at the promoter template strand will be determined by SELEX, gel mobility and NMR spectroscopy. The role of supercoiling and SSB in activation of N4 vRNAP promoter swill be determined by chemical reactivity, footprinting and kinetic analysis. We will characterize SSB mutants which specifically affect N4 early RNA synthesis to understand why SSB but not other single-stranded DNA binding proteins can activate N4 vRNAP promoters. The sequence of the vRNAP gene will be completed in order to manipulate it genetically to determine the domain of the protein responsible for promoter recognition. Finally, we will initiate studies to investigate the mechanism of transcription termination by N4 vRNAP. Middle N4 RNAs are synthesized by a second phage-coded, RNA polymerase (N4 RNAP II) composed of two polypeptides 30- and 40-kDa. A third 15-kDa protein allows N4 RNAPII to recognize its promoters which share sequence homology at two blocks present at variable distance from each other (t/aAAAT at +1 and Tt/aCTGGACa/t, 14-21 bp upstream). We will determine the relevance to promoter activity of the conserved sequences (and distance between them) at the sites of transcription initiation by deletion and mutation analysis. The interaction of the 15-kDa polypeptide with promoters will be characterized by gel retardation, footprinting and the isolation of change of specificity mutants. We will initiate structural studies on the 15-kDa polypeptide. We have recently shown that, although heterodimeric, N4 RNAP II shares sequence homology with the T7-like and yeast mitochondrial RNA polymerases and therefore, is the smallest member of this family. We will initiate structural and functional studies on N4 RNAP II. We will determine its interaction with the 15-kDa polypeptide at the promoter by footprinting and crosslinking analysis and the localization of the initial nucleotide binding site, DNA binding site and RNA binding using photocrosslinking nucleotide analogs.
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