REGULATORY PROTEIN-DNA INTERACTIONS -- PHYSICAL STUDIES

调节蛋白质-DNA 相互作用——物理研究

• 批准号: 3273064
• 负责人: Arnold Revzin
• 金额: $ 12.4万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1979
• 资助国家: 美国
• 起止时间: 1979-02-01 至 1987-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3273064
• 关键词: DNA; DNA directed RNA polymerase; Escherichia coli; affinity chromatography; bacterial genetics; density gradient ultracentrifugation; electron microscopy; enzyme complex; fluorescent dye /probe; gel electrophoresis; genetic transcription; lac operon; mutant; polynucleotides; thermodynamics

The goal of this research is to elucidate the molecular mechanisms involved in regulation of transcription, using as model systems two catabolite sensitive operons of E. coli. A variety of biophysical and biochemical methods will be applied to study interactions of the catabolite activator protein (CAP) and RNA polymerase with DNA fragments containing wild type or mutant promoter regions of the lactose and galactose operons. The techniques to be used include gel electrophoresis (for study of DNA-protein binding and dissociation as well as of transcription products), nuclease protection experiments which identify base sequences covered by DNA-bound proteins, centrifugation, electron microscopy, and optical methods such as fluorescence. Transcriptional initiation at the molecular level is more complex than originally thought. The gal promoter, for instance, is known to contain overlapping start sites for mRNA synthesis, one of which responds to CAP, the other not. Recent work on this project has shown that two CAP molecules are involved in stimulation of gal transcription. Likewise, while the lac operon shows a 1:1:1 stoichiometry for CAP:polymerase:promoter interactions, there is mounting evidence that CAP and RNA polymerase may interact in more than one way at the lac control region. The proposed research involves study of the biochemistry of these systems to characterize them and to clarify how these intricate regulatory mechanisms are modulated and fine-tuned in vivo. The techniques to be used (and developed as needed) will be applicable to study of specific, non-histone chromosomal proteins in mammalian organisms. The concepts derived from this work on a bacterial system will influence future research on control processes in eukaryotic cells. Ultimately, the philosophy underlying the project is that a detailed understanding of normal, regulatory processes is crucial to unraveling of the mysteries of uncontrolled, malignant cell growth and of other pathological conditions as well.

这项研究的目的是阐明涉及的分子机制 在调节转录时，使用AS AS模型系统两个分解代谢物 大肠杆菌的敏感操纵子。 多种生物物理和生化 方法将应用于研究分解代谢物激活剂的相互作用 蛋白质（CAP）和RNA聚合酶，带有含有野生型或的DNA片段 乳糖和半乳糖操纵子的突变启动子区域。 这 要使用的技术包括凝胶电泳（用于研究DNA蛋白 结合和解离以及转录产物），核酸酶 保护实验，识别由DNA结合覆盖的基础序列 蛋白质，离心，电子显微镜和光学方法（例如 荧光。 分子水平的转录启动比 最初想到。 例如，GAL启动子已知包含 MRNA合成的重叠起始位点，其中之一对CAP做出了反应， 另一个不是。 该项目的最新工作表明了两个帽子 分子参与刺激GAL转录。 同样地， lac操纵子显示1：1：1的化学计量法 上限：聚合酶：启动子相互作用，有越来越多的证据表明上限 RNA聚合酶可以在LAC控制下以多种方式相互作用 地区。 拟议的研究涉及研究这些的生物化学 表征它们并阐明这些复杂的调节的系统 机理在体内进行调节和微调。 要使用的技术（并根据需要开发）适用于 哺乳动物中特定的非启用染色体蛋白的研究 有机体。 从细菌系统上的这项工作中得出的概念将 影响对真核细胞控制过程的未来研究。 最终，该项目的哲学是一个详细的 了解正常的，监管过程对于解散 不受控制的，恶性细胞生长和其他的奥秘 病理状况。