Phage-induced modifications of RNA polymerase

描述（由申请人提供）：我们的长期目标是了解细胞RNA聚合酶（RNAP）的功能和调节的分子细节。噬菌体进化出复杂的机制来调节细菌宿主的转录以满足病毒的需要。噬菌体编码的转录调节因子的数量超过细菌调节因子的数量。噬菌体调节系统通常是紧凑的、稳健的和有效的。噬菌体诱导宿主RNAP修饰的研究提供了适用于细菌和高等生物的基因表达调控的重要范例。本研究的目的是研究噬菌体诱导的细菌宿主RNAP的修饰以及这些修饰在病毒发育中的作用。在体外，将鉴定噬菌体调节剂靶向的RNAP位点，并确定噬菌体调节剂的作用机制。在体内，遗传和基因组方法将用于了解噬菌体编码的抑制剂修饰RNAP的生物学后果。将分析以下模型系统： T4噬菌体。本研究将探讨终止因子Ale的分子机制。将确定T4 AsiA对宿主和早期病毒基因的负调节和对中期病毒基因的正调节的机制。将研究RNAP α的ADP-核糖基化在调节早期和中期病毒转录中的作用。 T7噬菌体。T7 gp0.7对RNAP β磷酸化的作用、E. coli RNAP转录的抑制作用，并研究gp 2在病毒DNA包装中的作用。 Sp 6噬菌体。Sp 6编码的S.鼠伤寒RNAP将被纯化和表征。 Xp 10噬菌体。一种新的Xp 10编码的抗终止因子p7的分子机制将被确定。全局转录谱分析将用于更好地理解Xp 10的基因表达策略，Xp 10是一种非常不寻常的噬菌体，似乎联合收割机结合了充分研究的T7和λ λ的调控模式。 本申请的主题统一性源于其对病毒发育期间通过共价修饰或RNAP相互作用蛋白对细菌RNAP的负调节的关注。对噬菌体编码蛋白和宿主RNAP修饰的研究将导致对病毒生物学的更深入理解。另一方面，噬菌体编码的转录抑制剂将被用作分子探针，以更好地了解RNAP机制和调控，并发现RNAP位点，可以为药物设计的目标。

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.