Phage-induced modifications of RNA polymerase

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DESCRIPTION (provided by applicant): Our long-term goal is to understand the function and regulation of cellular RNA polymerase (RNAP) in molecular detail. Bacteriophages evolved elaborate mechanisms to regulate transcription of bacterial host to serve viral needs. The number of phage-encoded transcription regulators exceeds the number of bacterial regulators by orders of magnitude. Phage regulatory systems are usually compact, robust and efficient. Studies of a handful of phage-induced modifications of host RNAP provided important paradigms of regulation of gene expression that are applicable to bacteria and higher organisms. The goal of this research is to study phage-induced modifications of bacterial host RNAP and the role of these modifications in viral development. In vitro, RNAP sites that are targeted by phage regulators will be identified and the mechanisms of action of phage regulators will be determined. In vivo, genetic and genomic approaches will be used to understand the biological consequences of RNAP modifications by phage-encoded inhibitors. The following model systems will be analyzed: The T4 phage. Molecular mechanism of termination factor Ale will be studied. The mechanism of negative regulation of host and early viral genes and positive regulation of middle viral genes by T4 AsiA will be determined. The role of ADP-ribosylation of RNAP alpha in regulation of early and middle viral transcription will be studied. The T7 phage. The role of RNAP beta phosphorylation by T7 gp0.7, the molecular mechanism of E. coli RNAP transcription inhibition by T7 gp2, and the role of gp2 in viral DNA packaging will be investigated. The Sp6 phage. Sp6-encoded inhibitor(s) of S. typhimurium RNAP will be purified and characterized. The Xp10 phage. Molecular mechanism of a novel Xp 10-encoded antitermination factor p7 will be identified. Global transcription profiling will be used to better understand gene expression strategy of Xp10, a highly unusual phage that appears to combine the regulatory paradigms of well-studied T7 and lambda phages. The thematic unity of this application stems from its focus on negative regulation of bacterial RNAP by covalent modifications or RNAP-interacting proteins during viral development. Studies of phage-encoded proteins and modifications of host RNAP will lead to deeper understanding of viral biology. On the other hand, phage-encoded transcription inhibitors will be used as molecular probes to better understand RNAP mechanism and regulation and to uncover RNAP sites that can be targets for drug design.