AS Mechanisms of RNA Polymerase-Promoter and lac Repressor-Operator Interactions

RNA 聚合酶启动子和 lac 抑制子-操纵子相互作用的 AS 机制

• 批准号: 9442919
• 负责人: M. THOMAS RECORD
• 金额: $ 0.8万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9442919
• 关键词: Active Sites; Amides; Antibiotics; Bacteria; Bacterial RNA; Betaine; Binding; Biochemical; Biological Assay; Biopolymers; Cations; Complex; Coupled; DNA; DNA-Directed RNA Polymerase; Data Analyses; Development; Elements; Equilibrium; Escherichia coli; Fluorescence Resonance Energy Transfer; Generations; Genes; Glycerol; Glycine; Goals; Hydrogen Bonding; Hydrophobicity; Individual; Kinetics; Label; Laboratories; Lac Repressors; Length; Methods; Modeling; Molecular Conformation; Nucleic Acids; Pathway interactions; Proline; Proteins; Radiolabeled; Regulation; Repression; Research; Role; Salts; Series; Sodium Chloride; Solubility; Source; Techniques; Testing; Thermodynamics; Transcription Initiation; Transcriptional Regulation; Trehalose; Urea; Variant; Virulent; Water; cyanine dye; design; functional group; novel; polyol; pressure; promoter; public health relevance; solute; stopped-flow fluorescence; transcription factor; vapor

 DESCRIPTION (provided by applicant): An immediate goal of this research is to determine the series of large conformational changes in E. coli σ70 RNA polymerase (RNAP) and promoter DNA that convert the initial specific (closed) complex to an unstable open complex and at some promoters subsequently stabilize this initial open complex. These conformational changes include large-scale bending, wrapping and opening of promoter DNA and hinge-bending, coupled folding and assembly of mobile elements of RNAP. An understanding of these conformational changes and their role in the initiation mechanism is necessary to understand regulation of initiation by promoter sequence and transcription factors, and to design new antibiotics. Techniques used in this laboratory to study the kinetics and characterize these conformational changes include fast footprinting and filter binding assays with radiolabeled DNA using rapid quench mixing, stopped flow fluorescence kinetic methods (FRET, PIFE) with cyanine-dye labeled DNA, and determination and interpretation of solute and salt effects on rate and equilibrium constants of mechanistic steps. Solute and salt effects on rate constants provide information about conformational changes and interactions in forming transition states, a source of mechanistic information not available by other methods. RNAP σ70 region 1 variants and promoter truncation variants are compared with wild-type RNAP and full-length promoters. We are testing the hypotheses that the rate of open complex formation is regulated by promoter-specific differences in the fraction of the ensemble of closed complexes that are sufficiently advanced to open, with the downstream duplex bent into the active site cleft. A second immediate goal of this research is to obtain the thermodynamic information on the interactions of key biochemical solutes with model compounds displaying protein and nucleic acid functional groups that is needed to interpret solute effects on rate and equilibrium constants and characterize transition states and intermediates. Vapor pressure osmometry and solubility assays are used to quantify preferential interactions of small solutes including urea and other amides, glycerol and other polyols, osmolytes including glycine betaine, proline, and trehalose, and the series of Hofmeister salts (from GuHSCN to Na2SO4) with model compounds displaying the functional groups of biopolymers. Novel analyses of these data are being used to quantify interactions of these solutes with the functional groups of nucleic acids and proteins, and interactions between individual functional groups. As tests of the use of solutes to determine mechanisms, solute effects on the kinetics of forming a lac repression complex and of opening and stabilizing the RNA polymerase (RNAP)- promoter initiation complex are being determined and interpreted in terms of mechanism. From determinations of group-group interactions, new quantitative information is obtained about the hydrophobic (C-C) effect, amideN-amideO hydrogen bonding, and cation-π, -CH-π and amideO-amideC (n-π*) interactions in water.

 描述（由适用提供）：这项研究的一个直接目标是确定大肠杆菌σ70RNA聚合酶（RNAP）和启动子DNA中的一系列大浓度变化，这些变化将初始特异性（封闭）复合物转换为不稳定的开放式复合物，并在某些启动子处于此最初的开放式复合物。这些组成变化包括启动子DNA和铰链弯曲的大规模弯曲，包裹和打开，耦合的折叠以及RNAP移动元件的组装。对这些构象变化及其在主动机制中的作用的理解对于了解启动子序列和转录因子对主动性的调节以及设计新的抗生素是必要的。该实验室中用于研究动力学和表征这些构象变化的技术包括使用快速淬灭的DNA进行快速的足迹和过滤器结合测定，使用快速淬火混合，停止流动荧光动力学方法（FRET，PIFE），用氰氨基型染色的DNA标记的DNA和固体和盐的确定和盐分对固体和盐的解释对速率和等效性的效应。溶质和盐对速率常数的影响提供了有关形成过渡状态中宪法变化和相互作用的信息，这是其他方法无法获得的机械信息来源。将RNAPσ70区域1变体和启动子截断变体与野生型RNAP和全长启动子进行比较。我们正在测试以下假设：开放式复合物的速率受启动子特异性差异的调节，在封闭复合物的集合的分数中，这些差异足以开放，而下游双工弯曲到活跃的位点裂缝中。这项研究的第二个直接目标是获取有关关键生化溶剂与模型化合物的相互作用的热力学信息，这些化合物显示蛋白质和核酸官能团，这是解释对速率和平衡常数的倾向影响的所需的 并表征过渡状态和中间体。蒸气压渗道测定法和溶液测定法用于量化小溶剂的优先相互作用，包括尿素和其他酰胺，甘油和其他多元醇，包括甘氨酸小甜菜蛋白，脯氨酸和天虫的渗透液，以及一系列HOFMEISTER SALES（从Guhscn到Na2SO4）（从GUHSCN到NA2SO4）与模型组合型组成的组合，以显示功能的组合。这些数据的新分析用于量化这些太阳能与核酸和蛋白质官能团的相互作用，以及各个官能团之间的相互作用。作为确定机制的使用的测试，正在用机制确定和解释对形成LAC表达复合物以及开放和稳定RNA聚合酶（RNAP）启动启动复合物的动力学的固体影响。从对群体相互作用的确定，可以获得有关疏水（C-C）效应，氨基氨基氢键以及阳离子-π，-CH-π和酰胺-Amidec（N-π*）相互作用的新定量信息。