Modifications of host RNA polymerase during bacteriophage development

描述（由申请人提供）：我们的长期目标是从分子上详细了解细菌DNA依赖性RNA聚合酶（RNAP）的功能和调节。细菌病毒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。它们进化出的调节机制的种类和数量远远超过了细菌调节机制的种类。噬菌体调节系统是紧凑的、稳健的和有效的（即，噬菌体编码的蛋白质很小，它们与宿主RNAP紧密相互作用，并且它们的调节作用很强）。研究表明，只有少数宿主RNAP被感染的细菌（如大肠杆菌）编码的蛋白质修饰。大肠杆菌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，噬菌体蛋白调节RNAP功能的结构理解通常是缺乏的。本研究的目标是i）鉴定由热释光蛋白编码的蛋白质，（栖热菌属的非感染性细菌），其与宿主RNAP结合; ii）确定这些蛋白质的结合位点及其结合的功能后果，以及iii）与主要结构组合作，确定噬菌体蛋白质与栖热菌RNAP之间的复合物的结构，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。所提出的策略允许，第一次，直接与RNAP结合转录因子的功能和它们与靶酶的复合物的结构。新的噬菌体编码的转录调节因子与RNAP的不同亚基相互作用，并影响转录周期的不同阶段的详细表征将提供新的分子探针，以更好地了解RNAP机制和调节，并发现RNAP网站，可以为药物设计的目标。只要有可能，将确定结合宿主RNAP的热噬菌体蛋白在病毒发育中的作用。 公共卫生相关性：在细菌病毒感染期间，细菌宿主的基因转录酶- RNA聚合酶（RNAP）-停止表达宿主基因并开始表达病毒基因;这种变化通常是由噬菌体蛋白与宿主RNAP结合引起的。我们建议识别和表征，在功能上和结构上，几个噬菌体蛋白，结合和改变RNAP的活性从栖热菌，唯一的细菌RNAP可以结晶，其结构是已知的。研究结果将首次直接关联转录调节因子的功能和结构，从而更好地理解细菌转录，并帮助设计抑制细菌RNAP的新化合物，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.