Modifications of host RNA polymerase during bacteriophage development

描述（由申请人提供）：我们的长期目标是了解细菌dna依赖性RNA聚合酶（RNAP）的分子细节功能和调控。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的调节机制的数量大大超过了细菌调节机制的多样性。噬菌体调控系统紧凑、稳健、高效（即噬菌体编码的蛋白体积小，与宿主RNAP相互作用紧密，调控作用强）。通过噬菌体编码的蛋白质感染被充分研究的细菌（如大肠杆菌），仅对宿主RNAP进行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，对噬菌体蛋白调控RNAP功能的结构理解普遍缺乏。本研究的目的是1)鉴定与宿主RNAP结合的热噬细胞（感染热菌属细菌的噬菌体）编码的蛋白质；ii)确定这些蛋白质的结合位点和它们结合的功能后果，iii)与领先的结构小组合作，确定噬菌体蛋白和Thermus RNAP之间复合物的结构，Thermus RNAP是唯一形成衍射晶体的细菌RNAP，并且可以获得高分辨率的结构信息。所提出的策略首次允许将rnap结合转录因子的功能及其复合物的结构与靶酶直接联系起来。详细表征新的噬菌体编码转录调节剂与RNAP的不同亚基相互作用并影响转录周期的不同阶段，将提供新的分子探针，以更好地了解RNAP的机制和调控，并揭示可作为药物设计靶点的RNAP位点。只要有可能，将确定结合宿主RNAP的热噬蛋白在病毒发育中的作用。

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.