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（由申请人提供）：本研究的直接目标是确定E. coli σ70 RNA聚合酶（RNAP）和启动子DNA，其将初始特异性（闭合）复合物转化为不稳定的开放复合物，并且在某些启动子处随后稳定该初始开放复合物。这些构象变化包括启动子DNA的大规模弯曲、缠绕和打开以及RNAP的移动的元件的铰链弯曲、偶联折叠和组装。了解这些构象变化及其在起始机制中的作用对于理解启动子序列和转录因子对起始的调节以及设计新的抗生素是必要的。本实验室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混合的放射性标记DNA的快速足迹法和过滤器结合试验，使用花青染料标记DNA的停流荧光动力学方法（FRET，PIFE），以及确定和解释溶质和盐对机械步骤的速率和平衡常数的影响。溶质和盐对速率常数的影响提供了有关形成过渡态的构象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的来源。将RNAP σ70区域1变体和启动子截短变体与野生型RNAP和全长启动子进行比较。我们正在测试的假设，开放的复合物形成的速度是由启动子特定的差异调节的分数的合奏的封闭复合物，是足够先进的开放，与下游双链体弯曲成活性位点裂缝。本研究的第二个直接目标是获得关键生化溶质与显示蛋白质和核酸官能团的模型化合物相互作用的热力学信息，这是解释溶质对速率和平衡常数的影响所必需的 并表征过渡态和中间体。蒸气压渗透压法和溶解度测定用于定量小溶质包括尿素和其他酰胺，甘油和其他多元醇，渗透剂包括甘氨酸甜菜碱，脯氨酸和海藻糖，和Hofmeister盐系列（从GuHSCN到Na 2SO 4）与显示生物聚合物官能团的模型化合物的优先相互作用。这些数据的新分析正在被用来量化这些溶质与核酸和蛋白质的官能团的相互作用，以及各个官能团之间的相互作用。作为使用溶质来确定机制的测试，溶质对形成乳糖阻遏复合物以及打开和稳定RNA聚合酶（RNAP）-启动子起始复合物的动力学的影响正在被确定并在机制方面被解释。通过基团间相互作用的测定，获得了有关水中疏水（C-C）效应、酰胺N-酰胺O氢键以及阳离子-π、-CH-π和酰胺O-酰胺C（n-π*）相互作用的新的定量信息。