Phage-induced modifications of RNA polymerase

描述(申请人提供)：我们的长期目标是了解细胞RNA聚合酶(RNAP)的功能和调控的分子细节。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子的数量比细菌调节子的数量多出几个数量级。噬菌体调控系统通常紧凑、健壮和高效。对少数噬菌体诱导的宿主RNAP修饰的研究为适用于细菌和高等生物的基因表达调控提供了重要的范例。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展中的作用。在体外，将确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被用来理解噬菌体编码的抑制物对RNAP修饰的生物学后果。我们将分析以下模型系统： T4噬菌体。终止因子ALE的分子机制有待进一步研究。T4 Asia对宿主和早期病毒基因的负调控和对中病毒基因的正调控的机制将被确定。RNAPα的ADP核糖化在调节病毒早期和中期转录中的作用将被研究。 T7噬菌体。我们将研究T7 gp0.7对RNAPβ的磷酸化作用，T7 gp2抑制大肠杆菌RNAP转录的分子机制，以及gp2在病毒DNA包装中的作用。 Sp6噬菌体。鼠伤寒沙门氏菌RNAP的SP6编码抑制物(S)将被提纯和鉴定。 Xp10噬菌体。一种新的XP10编码的抗终止因子p7的分子机制将被确定。全球转录图谱将被用来更好地理解Xp10的基因表达策略，Xp10是一种非常不寻常的噬菌体，似乎结合了研究充分的T7和lambda噬菌体的调控范式。 这一应用的主题统一性源于它关注在病毒发育过程中通过共价修饰或RNAP相互作用蛋白对细菌RNAP的负调控。对噬菌体编码蛋白的研究和对宿主RNAP的修饰将有助于加深对病毒生物学的理解。另一方面，噬菌体编码的转录抑制物将被用作分子探针，以更好地了解RNAP的机制和调控，并发现可能成为药物设计靶点的RNAP位点。

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.