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大肠杆菌 σ70 RNA 聚合酶 (RNAP) 和启动子 DNA 中一系列大的构象变化，这些变化将最初的特异性（闭合）复合物转化为不稳定的开放复合物，并且这些构象变化包括启动子 DNA 的大规模弯曲、包裹和打开以及 RNAP 移动元件的铰链弯曲、耦合折叠和组装。它们在启动机制中的作用对于了解启动子序列和转录因子对启动的调节以及设计本实验室用于研究动力学和表征这些构象变化的技术（包括放射性标记 DNA 的快速足迹和过滤结合测定）是必要的。使用快速淬灭混合、停流荧光动力学方法（FRET、PIFE）以及花青染料标记的 DNA，并测定和解释溶质和盐对机械步骤的速率和平衡常数的影响。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制信息来源，我们将 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。正在测试以下假设：开放复合物形成的速率是由启动子特异性差异调节的，其中封闭复合物整体的比例足够先进以打开，下游双链体弯曲到活性位点裂缝中。这项研究的目的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，这些信息是解释溶质对速率和平衡常数的影响所需的 并表征过渡态和中间体，蒸气压渗透压和溶解度测定用于量化小溶质（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）以及霍夫迈斯特盐系列的优先相互作用。从 GuHSCN 到 Na2SO4）以及显示生物聚合物官能团的模型化合物正在用于对这些数据进行新颖的分析。量化这些溶质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，作为使用溶质来确定机制的测试，溶质对形成 lac 抑制复合物以及打开和稳定 lac 抑制复合物的动力学的影响。 RNA 聚合酶 (RNAP)-启动子起始复合物正在确定并从机制方面进行解释，从基团间相互作用的测定中，获得了有关疏水 (C-C) 效应、酰胺 N-酰胺 O 氢键、以及水中的阳离子-π、-CH-π 和酰胺O-酰胺C (n-π*) 相互作用。