Structure/Function of Transcription Complex Regulation

描述（由申请人提供）：本项目的长期目标是确定转录延伸复合物内的相互作用，以及与RNA聚合酶控制RNA合成的调节蛋白和核蛋白复合物的相互作用。RNA聚合酶的暂停和过早终止在许多方面影响细菌基因的表达。转录核蛋白模板的结构是这些调控事件的重要组成部分。在某些情况下，模板的核蛋白结构导致需要专门的调节蛋白修饰转录复合物以使其抵抗暂停和终止。暂停和终止的基本机制以及调节蛋白控制暂停和终止的机制都依赖于延伸复合物内相互作用的变化，但这些变化目前还知之甚少。这些相互作用的中心目标是将RNA聚合酶中的触发环折叠成触发螺旋，这是催化RNA合成所需的。RNA聚合酶中的这些调节事件与酶与核蛋白模板的接触之间的协同作用甚至更不清楚。为了理解这种协同作用，必须确定细菌细胞中核蛋白模板的组成和结构。通过与质谱专家的合作，将开发一种基于序列特异性DNA捕获和定量蛋白质组学的新方法，以分析核蛋白模板的组成和结构，确定核蛋白复合物在不同环境条件下的变化，并确定核蛋白复合物在与转录RNA聚合酶相互作用时如何变化。已知细菌类核中核蛋白复合物结构的变化在通过侧向基因转移获得的外源DNA沉默、细菌发病机制和细菌应激反应中起关键作用。因此，该项目将 对人类健康的关键主题产生广泛影响，从人类微生物组的基因流动到微生物发病机制。它还将有助于发展一种重要的新技术，即DNA序列靶向无偏蛋白质组学，该技术在生物技术、人类医学以及原核和真核分子生物学中具有广泛的应用。

DESCRIPTION (provided by applicant): The long-term goal of this project is to define the interactions within transcription elongation complexes and with regulatory proteins and nucleoprotein complexes that govern RNA synthesis by RNA polymerase. Pausing and premature termination by RNA polymerase affect expression of bacterial genes in many ways. The structure of the nucleoprotein template for transcription is an important component of these regulatory events. In some cases, the nucleoprotein structure of the template causes a requirement that specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Both the basic mechanisms of pausing and termination and the mechanisms by which regulatory proteins control pausing and termination depend on changes to interactions within the elongation complex that are poorly understood. A central target of these interactions is folding of the trigger loop in RNA polymerase into the trigger helices, which is required to catalyze RNA synthesis. The synergy between these regulatory events in RNA polymerase and the enzyme's contacts to the nucleoprotein template are even less well understood. To understand this synergy, the composition and structure of the nucleoprotein template in bacterial cells must be defined. Through collaboration with an expert in mass spectrometry, a new approach based on sequence-specific DNA capture and quantitative proteomics will be developed to analyze the composition and structure of the nucleoprotein template, to identify changes in nucleoprotein complexes in different environmental conditions, and to identify how nucleoprotein complexes change when interacting with transcribing RNA polymerase. Changes in the structure of nucleoprotein complexes in the bacterial nucleoid are known to play key roles in silencing of foreign DNA acquired by lateral gene transfer, in bacterial pathogenesis, and in bacterial stress responses. Thus, the project will have broad impact on key topics in human health ranging from gene flow in the human microbiome to microbial pathogenesis. It also will contribute to the development of an important new technology of DNA sequence-targeted unbiased proteomics with broad applications in biotechnology, human medicine, and both prokaryotic and eukaryotic molecular biology.