Transcription regulation through RNAP secondary channel

通过 RNAP 二级通道进行转录调控

• 批准号: 7087923
• 负责人: DMITRY G VASSYLYEV
• 金额: $ 64.96万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7087923
• 关键词: DNA directed RNA polymerase; X ray crystallography; bacterial RNA; bacterial genetics; crosslink; gene expression; genetic transcription; protein protein interaction; transcription factor

DESCRIPTION (provided by applicant): RNA polymerase is the central enzyme of gene expression in all domains of life. Changes in gene expression patterns punctuate the progress of normal and neoplastic development of eukaryotes and onset of virulence in bacteria. One of the most exciting goals in transcription studies now is identification of the 'hot spots' on RNA polymerase that mediate its interactions with regulators; these sites could serve as targets for design of the bacterial RNA polymerase inhibitors. Recent structural studies of large, ~450 kDa multi-subunit bacterial RNA polymerases revealed a highly intricate architecture of the enzyme. Various cavities and channels on the enzyme surface that provide access to the functionally important protein regions and to the nucleic acid chains likely serve as targets for the regulatory transcription factors. Among these targets, the RNA polymerase substrate entry (secondary) channel has been recently recognized as a binding site for an ever-growing number of transcription regulators. Determinants in the secondary channel mediate stringent response of bacteria to stress and starvation, provide tight homeostatic control of the rRNA expression, and underlie the 3 known activities of RNA polymerase: nucleotide addition, endo- and exo-nucleolytic cleavages of the nascent RNA, and in turn likely control the fidelity of transcription. The goal of this project is to structurally and functionally characterize the secondary channel as one of the crucial regulatory sites in RNA polymerase to aid in understanding of gene regulation and design of novel antibacterial compounds. Specific aims of this study will be as follows. First, to elucidate the structural basis of the mechanism by which transcript cleavage factors 'rescue' arrested transcription complexes. Structures of RNA polymerase elongation complexes with bound transcript cleavage factors GreA, GreAl, GreA2 and GreB will be determined at the atomic level using the nucleic acid scaffolds that mimic the backtracked elongation complexes, their native substrates. Second, to provide further insights into regulation of the rRNA synthesis and the molecular mechanism of stringent response, structure of the RNA polymerase with bound major regulator, alarmone ppGpp, and its protein co-regulator DksA, will be determined. Third, a combination of biochemical and genetic approaches (structure-guided mutagenesis, in vitro transcription assays, crosslinking, etc) will be used to dissect the determinants in RNA polymerase, nucleic acids, and transcription factors that mediate control of transcription through the secondary channel.

描述（由申请人提供）：RNA聚合酶是生命所有领域中基因表达的中心酶。基因表达模式的变化突出了真核生物正常和肿瘤发育的进展以及细菌中毒力的发作。现在，转录研究中最令人兴奋的目标之一是鉴定RNA聚合酶的“热点”，该聚合酶介导了其与调节剂的相互作用。这些位点可以用作设计细菌RNA聚合酶抑制剂的靶标。大型大型结构研究，〜450 kDa多生细菌RNA聚合酶揭示了该酶的高度复杂结构。酶表面上的各种空腔和通道可提供对功能上重要的蛋白质区域和核酸链的访问，这可能是调节转录因子的靶标。在这些靶标中，RNA聚合酶底物进入（辅助）通道最近被认为是不断增长的转录调节剂的结合位点。次级通道中的决定因素介导细菌对压力和饥饿的严格反应，对RRNA表达提供了严格的稳态控制，并构成了RNA聚合酶的3种已知活性：核苷酸添加，内核和外核核解液化液NASCENT RNA的裂解，并反过来控制转录的富裕度。该项目的目的是在结构和功能上将次级通道作为RNA聚合酶中关键的调节位点之一，以帮助理解新型抗菌化合物的基因调节和设计。这项研究的具体目标将如下。首先，为了阐明转录本裂解因子“救援”逮捕的转录复合物的结构基础。 RNA聚合酶伸长率复合物的结构使用结合的转录裂解因子Grea，Greal，Grea2和Greb将在原子水平上使用模仿其本地底物的回溯延伸延伸复合物的核酸支架来确定原子水平。其次，将确定将确定对RRNA合成的调节和严格响应的分子机制，RNA聚合酶与结合主要调节剂的结构，Alarmone PPGPP及其蛋白质共调节剂DKSA。第三，将使用生化方法和遗传方法（结构引导的诱变，体外转录测定，交联等）的结合，以剖析RNA聚合酶，核酸，核酸和转录因子的决定因素，从而通过二级通道介导转录控制。