REGULATORY PROTEIN-DNA INTERACTIONS--PHYSICAL STUDIES

调节蛋白质-DNA 相互作用——物理研究

基本信息

批准号：
3273071
负责人：
Arnold Revzin
金额：
$ 16.68万
依托单位：
MICHIGAN STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1979
资助国家：
美国
起止时间：
1979-02-01 至 1993-12-31
项目状态：
已结题

项目摘要

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 physical and biochemical methods will be applied to study interactions of purified RNA polymerase and catabolite activator protein (CAP) 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 the study of DNA-protein binding and for analysis of transcription products), site-directed mutagenesis, nuclease protection experiments, centrifugation, and fluorescence. The project is specifically aimed at study of three issues of current interest in transcriptional control: i. What are the characteristics and roles of the overlapping RNA polymerase binding sites found at many promoters? ii. What is the function of multiple copies of regulatory proteins which may interact at the promoter region? Two CAP molecules are known to bind to the gal promoter. How do these interact with DNA, with RNA polymerase, and with each other to facilitate initiation of mRNA synthesis? Does CAP work in different ways at different operons? iii. Are there interactions between CAP molecules (or between CAP and RNA polymerase) bound to nonadjacent sites on DNA? If so, how do these lead to enhancement of transcription? The techniques to be used (and developed as needed) will be applicable to study of specific chromosomal proteins in mammalian organisms. The concepts which emerge from this work will influence future research on control processes in eukaryotic cells. For example, enhancer sequences in eukaryotes may function through direct contacts between proteins bound to different regions of DNA. Ultimately, the philosophy underlying this project is that a detailed understanding of normal regulatory processes is crucial to unravelling the mysteries of uncontrolled, malignant cell growth and of other pathological conditions as well.

这项研究的目的是阐明分子机制参与转录调节，使用AS模型系统两个大肠杆菌的分解代谢物敏感操纵子。各种身体和生化方法将应用于研究纯化的RNA聚合酶和分解代谢物活化剂蛋白（CAP），带有与包含野生型或突变启动子区域的DNA片段乳糖和半乳糖操纵子。要使用的技术包括凝胶电泳（用于研究DNA蛋白结合以及用于转录产品的分析），定向诱变，核酸酶保护实验，离心和荧光。该项目专门针对研究的三个问题当前对转录控制的兴趣：我。重叠的RNA的特性和角色是什么在许多启动子中发现聚合酶结合位点？ ii。多个调节蛋白副本的功能是什么哪个可能在启动子区域相互作用？两个帽分子是已知可以与GAL启动子结合。这些如何与DNA相互作用，使用RNA聚合酶，彼此之间有助于启动 mRNA合成？帽子在不同的方式上以不同的方式工作操纵子？ iii。盖分子之间是否存在相互作用（盖之间和RNA聚合酶）与DNA上的非相邻位点结合？如果是这样，怎么样这些会导致转录的增强吗？要使用的技术（并根据需要开发）将是适用于哺乳动物中特定染色体蛋白的研究有机体。从这项工作中出现的概念将影响对真核细胞控制过程的未来研究。为了例如，真核生物中的增强子序列可以通过与DNA不同区域结合的蛋白质之间的直接接触。最终，这个项目的哲学是对正常调节过程的详细了解至关重要揭开不受控制的恶性细胞生长的奥秘以及其他病理状况。