Modifications of host RNA polymerase during bacteriophage development

描述(申请人提供)：我们的长期目标是了解细菌DNA依赖的RNA聚合酶(RNAP)的功能和调控的分子细节。细菌病毒--噬菌体--进化出复杂的机制来调节宿主的转录，以使其满足病毒的需要。噬菌体的多样性和它们进化出的调控机制的数量远远超过了细菌调控机制的多样性。噬菌体调控系统紧凑、健壮和高效(即噬菌体编码的蛋白很小，它们与宿主RNAP紧密相互作用，它们的调控效果很强)。对感染大肠杆菌等研究充分的细菌的噬菌体编码的蛋白质对宿主RNAP的少数修饰的研究，提供了适用于细菌和高等生物的基因表达调控的范例。然而，对噬菌体蛋白调节RNAP功能的结构了解普遍缺乏。这项研究的目标是：i)鉴定由嗜热菌(Thermus属细菌的噬菌体)编码的与宿主RNAP结合的蛋白质；ii)确定这些蛋白质的结合部位及其结合的功能后果；以及iii)与主要结构基团合作，确定噬菌体蛋白与Thermus RNAP之间的复合体的结构，Thermus RNAP是唯一形成衍射晶体的细菌RNAP，其高分辨率结构信息是可用的。所提出的策略首次允许将RNAP结合转录因子的功能及其与靶酶的复合体的结构直接联系起来。新的噬菌体编码的转录调控因子与RNAP的不同亚单位相互作用并影响转录周期的不同阶段，其详细特征将为更好地了解RNAP的机制和调控以及揭示可作为药物设计靶点的RNAP位点提供新的分子探针。只要有可能，就会确定与宿主RNAP结合的噬菌体蛋白在病毒发展中的作用。 公共卫生相关性：在被细菌病毒(噬菌体)感染期间，细菌宿主的基因转录酶--RNA聚合酶(RNAP)--停止表达宿主基因，而开始表达病毒基因；这种变化通常是由噬菌体蛋白与宿主RNAP结合引起的。我们建议从功能和结构上鉴定和鉴定几种与Thermus细菌RNAP结合并改变RNAP活性的噬菌体蛋白，Thermus细菌RNAP是唯一可以结晶且结构已知的细菌RNAP。这一结果将首次使转录调控因子的功能和结构直接相关，有助于更好地了解细菌转录，并有助于设计新的化合物来抑制细菌RNAP，而细菌RNAP是抗生素的有效靶点。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the function and regulation of bacterial DNA-dependent RNA polymerase (RNAP) in molecular detail. Bacterial viruses--phages--evolved elaborate mechanisms to regulate host transcription in order to make it serve the needs of the virus. The variety of phages and the number of regulatory mechanisms that they evolved vastly exceeds the variety of bacterial regulatory mechanisms. Phage regulatory systems are compact, robust, and efficient (i.e., phage-encoded proteins are small, they interact with host RNAP tightly, and their regulatory effects are strong). Studies of only a handful of modifications of host RNAP by proteins encoded by phages infecting well-studied bacteria such as E. coli provided paradigmatic examples of regulation of gene expression that are applicable to both bacteria and higher organisms. However, the structural understanding of regulation of RNAP function by phage proteins is generally lacking. The goal of this research is i) to identify proteins encoded by thermophages (phages infecting bacteria of the Thermus genus) that bind to host RNAP; ii) to determine the binding sites of these proteins and functional consequences of their binding, and iii) in collaboration with leading structural groups, to determine the structures of complexes between phage proteins and Thermus RNAP, the only bacterial RNAP that forms diffracting crystals and for which high-resolution structural information is available. The proposed strategy allows, for the first time, to directly relate the function of RNAP-binding transcription factors and the structure of their complexes with the target enzyme. Detailed characterization of new phage-encoded transcription regulators that interact with different subunits of RNAP and affect different stages of the transcription cycle will provide novel molecular probes to better understand RNAP mechanism and regulation and to uncover RNAP sites that can be targets for drug design. Whenever possible, the role of thermophage proteins that bind host RNAP in viral development will be determined. PUBLIC HEALTH RELEVANCE: During infection by bacterial viruses (phages) the gene transcription enzyme of bacterial host -- RNA polymerase (RNAP) -- stops expressing host genes and starts expressing viral genes; this change is often caused by the binding of phage proteins to host RNAP. We propose to identify and characterize, both functionally and structurally, several phage proteins that bind to and change the activity of RNAP from Thermus bacteria, the only bacterial RNAP which can be crystallized and for which the structure is known. The results will allow, for the first time, to directly relate the function and structure of transcription regulators, lead to better understanding of bacterial transcription and help design new compounds that inhibit bacterial RNAP, a validated target of antibiotics.