INTER & INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

描述（由申请人提供）：转录起始是细胞过程调节的主要点。本申请的长期目标是获得转录起始中涉及的所有步骤的详细理解，转录起始是一个过程，其中DNA依赖性酶RNA聚合酶首先定位启动子，并在随后的异构化步骤中在转录起始点附近熔化DNA双链体的片段以暴露DNA的模板链。在分子水平上理解转录对于推进这一基本细胞过程的基础知识将是重要的。此外，由于细菌RNA聚合酶之间的结构和功能特性的显著保守性，细菌转录机制是药物发现的有吸引力的靶标。因此，了解细菌转录将有助于发现新的抗生素，这是一个重要的健康相关问题，因为耐药微生物的问题越来越多。该建议的重点是E。大肠杆菌RNA聚合酶是一种亚基，在最近的研究中被证明在转录起始的所有步骤中起着密切的作用。将追求解决关于该亚基在通过RNA聚合酶发现和解链启动子DNA中的作用的基本问题的三个目标：目标#1。探讨转录泡形成起始的分子机制。目标2。确定σ区4 -β亚基“瓣”结构域接触的功能作用。目标3。确定sigma - DNA接触对于RNA聚合酶搜索启动子的相对重要性. 转录起始的分子事件是复杂的，并且涉及大的多组分复合物。因此，将使用结合生物物理学（荧光和质谱）、生物化学和分子生物学方法的多学科方法。预计将获得对RNA聚合酶功能中几种蛋白质-蛋白质和蛋白质-DNA接触的作用的详细了解。

DESCRIPTION (provided by applicant): Transcription initiation is a major point of regulation of cellular processes. The long-term goal of this application is to obtain a detailed understanding of all steps involved in initiation of transcription, a process in which DNA-dependent enzyme RNA polymerase first locates promoter and in subsequent isomerization step melts a segment of DNA duplex in the vicinity of transcription start point to expose the template strand of DNA. Understanding transcription at a molecular level will be important for advancing the basic knowledge of this fundamental cellular process. Additionally, bacterial transcription machinery is an attractive target for drug discovery due to a remarkable conservation of structural and functional properties among bacterial RNA polymerases. Understanding bacterial transcription will thus aid in discovery of new antibiotics, an important health related issue due to the increasing problems with drug resistant microorganisms. The proposal is focused on E. coli RNA polymerase a subunit that in recent studies was shown to play intimate roles in all steps of transcription initiation. Three aims addressing fundamental issues concerning the role of this subunit in finding and melting of promoter DNA by RNA polymerase will be pursued: Aim #1. To determine molecular mechanism of initiation of transcription bubble formation. Aim #2. To determine functional roles of sigma region 4 - beta subunit "flap" domain contact. Aim #3. To determine the relative importance of sigma - DNA contacts for promoter search by RNA polymerase. Molecular events of transcription initiation are complex and involve large multi component complexes. Thus, a multi disciplinary approach combining biophysical (fluorescence and mass spectroscopy), biochemical, and molecular biology methods will be used. It is expected that a detailed understanding of the role of several protein-protein and protein-DNA contacts in RNA polymerase function will be obtained.