ION EFFECTS ON INTERACTIONS OF RNA POLYMERASE AND DNA

离子对 RNA 聚合酶和 DNA 相互作用的影响

• 批准号: 3271644
• 负责人: M. THOMAS RECORD
• 金额: $ 27.36万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 1992-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3271644
• 关键词: DNA; DNA directed RNA polymerase; Escherichia coli; acidity /alkalinity; analytical ultracentrifugation; anions; binding proteins; conformation; enzyme structure; fluorescence spectrometry; gene expression; genetic promoter element; genetic regulation; genetic transcription; ionic strengths; lac operon; nucleic acid sequence; nucleoproteins; protein structure function; radionuclide double label; thermodynamics; tritium

The long-term objectives of this research are a) to determine the general biophysical principles involved in specificity and stability of protein-DNA complexes, and b) to apply these principles to understand the thermodynamic and kinetic basis of regulation of transcription initiation at prokaryotic promoters. Our fundamental biophysical strategy is to use ions and other physical variables to investigate the effects of changes in DNA recognition sequence on the thermodynamics and mechanisms of site- specific protein-DNA interactions, as well as to investigate important facilitating or competing multiple equilibria in these systems. Our major specific aims are 1) Kinetics and Mechanisms of RNA Polmerase-Promoter Interactions. Fluorescence-detected abortive initiation (FDAI) and filter-binding experiments on the strong lambda PR promoter and the weak lambda PRMupl promoter are proposed to characterize the intermediates on the pathway to open- complex formation and to determine the mechanistic steps affected by physical and chemical variables (supercoiling, temperature, pH, salt, effectors). Experiments on a family of promoter sequences spanning the range between the weak promoter lambda PRMupl and the strong consensus promoter sequence are proposed, in order to relate changes in DNA sequence and functional groups to individual mecha- nistic steps. 2) Thermodvnamics of lac Repressor-lac Operator Interactions. The thermodynamic origins of specificity and stability of binding of lac repressor to natural {Oc} and synthetic variants of the lac operator site are being examined by filter binding. Questions that will be addressed include our hypothesis of adaptability in recognition, the origin of the entropic driving force, and the question of whether noncovalent contacts provide independent (additive) or context-dependent non-additive) contributions to the binding free energy. This research will lead to a more detailed understanding of the relationship between structure {DNA sequence, protein conformation and composition} and function of two now-classical gene regulatory systems in vitro. Both the thermodynamic and mechanistic questions that we pose and the strategies (e.g. use of physical variables, expecially T, pH, ion concentrations) that we use to answer them are of general importance and applicability, so this work will serve as a model for studies on other systems. Since these regulatory systems can be studied at a quantitative level in vivo, our in vitro conclusions can be tested for physiological relevance.

本研究的长期目标是 a) 确定 涉及特异性和的一般生物物理学原理 蛋白质-DNA 复合物的稳定性，以及 b) 应用这些 了解热力学和动力学基础的原理 原核生物启动子转录起始的调控。 我们的基本生物物理策略是使用离子和其他 研究 DNA 变化影响的物理变量 热力学和位点机制的识别序列 特定的蛋白质-DNA 相互作用，以及研究 在这些方面重要的促进或竞争多重平衡 系统。我们的主要具体目标是 1) 动力学和机制 RNA 聚合酶-启动子相互作用。 荧光检测 流产启动（FDAI）和过滤器结合实验 强 lambda PR 启动子和弱 lambda PRMupl 启动子是 提议表征开放途径中的中间体 复杂的形成并确定受影响的机械步骤 通过物理和化学变量（超螺旋、温度、pH、 盐、效应器）。 启动子序列家族的实验 跨越弱启动子 lambda PRMupl 和 提出了强共有启动子序列，以便将 DNA序列和功能组对个体机械的改变 精妙的步骤。 2) lac Repressor-lac Operator的热力学 互动。 特异性和热力学起源 lac 阻遏物与天然 {Oc} 和合成物结合的稳定性 过滤器正在检查 lac 操作员站点的变体 绑定。 将要解决的问题包括我们的假设 识别的适应性，熵驱动的起源 力，以及非共价接触是否提供的问题 独立（相加）或上下文相关的非相加） 对结合自由能的贡献。 这项研究将使人们更详细地了解 结构{DNA序列、蛋白质构象之间的关系 两个现在经典的基因调控的组成}和功能 体外系统。 热力学和机械问题 我们提出的策略和策略（例如使用物理变量， 特别是 T、pH、离子浓度），我们用它们来回答它们 具有普遍的重要性和适用性，因此这项工作将 作为其他系统研究的模型。 由于这些 可以在体内定量研究调节系统， 我们的体外结论可以进行生理相关性测试。