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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

基本信息

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
项目类别：
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

项目摘要

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.