RNA POLYMERASE II AND THE TRANSCRIPTION COMPLEX

在转录水平上调控基因表达是调控基因表达的重要手段之一。 调节细胞生长和分化的主要手段。 异常 转录调控在肿瘤发生中起重要作用， 转型 尽管有大量的研究活动， 转录调控机制，我们才刚刚开始了解 多个转录因子的图片以及它们如何促进， 激活和抑制特定的转录。 目前， 已知RNA聚合酶内亚基相互作用的细节 这些亚基如何与蛋白质相互作用并受蛋白质的调节， 许多转录因子。 我们将专注于结构， 转录机制的功能和调节，强调 RNA聚合酶II上。 我们将研究RNA聚合酶II亚基 组织：通过识别稳定的蛋白质，通过蛋白质-蛋白质 交联，并通过合作的三维结构测定， 酵母酶 我们将确定哪些RNA聚合酶II亚基 参与与一般转录因子的相互作用， 溶液中的因子-聚合酶复合物和再起始复合物 在最小启动子系统（包括人IgH启动子， RNA聚合酶II和最基本的通用转录因子 TBP、TFIIB和RAP 30）。 我们将扩展我们最近的结果， RNA最大亚基C端结构域的重要性 聚合酶II，并确定与该结构域相互作用的因子。 虽然我们最终对人类转录调控感兴趣， 我们也将广泛使用酵母，因为这种上级 遗传学及其丰度和替代人类聚合酶的能力 在提取物和纯化系统中体外转录期间。 这 工作将建立在我们的优势，经验和以前的工作， RNA聚合酶的鉴定、纯化和表征， 转录因子 我们将严重依赖蛋白质， 蛋白质-DNA交联;关于使用单克隆抗体检测， 抑制和免疫纯化转录器的部分;以及 用于从凝胶洗脱的蛋白质的有效复性的改进方法， 印迹到膜上，或从溶解的包涵体。 我们相信，对RNA聚合酶II进行基础的、彻底的研究， 结构和与转录因子和DNA的关键相互作用将 为特异性转录机制提供了重要的新见解 以及转录是如何被控制的 长期目标是 研究是利用我们对特定聚合酶的详细了解- 转录因子的相互作用来设计干扰转录因子的试剂。 异常的调节相互作用对维持肿瘤的 状态

Regulation of gene expression at the level of transcription is one of the major means of regulating cell growth and differentiation. Abnormal transcriptional regulation plays an important role in neoplastic transformation. Despite a great amount of research activity on the mechanisms of transcriptional regulation, we are only beginning to get a picture of the multiple transcription factors and how they promote, activate, and repress specific transcription. Very little is presently known about the details of the subunit interactions within RNA polymerase II itself of how these subunits interact with and are modulated by the numerous transcription factors. We will concentrate on the structure, function and regulation of the transcription machinery, with an emphasis on RNA polymerase II. We will study the RNA polymerase II subunit organization: by identifying stable subassemblies, by protein-protein crosslinking, and by collaborating on the 3-D structure determination of the yeast enzyme. We will determine which RNA polymerase II subunits are involved in interactions with the general transcription factors both in factor-polymerase complexes in solution and in reinitiation complexes formed on a minimal promoter system (involving the human IgH promoter, RNA polymerase II and the most essential general transcription factors TBP, TFIIB, and RAP30). We will extend our recent results on the importance of the C-terminal domain of the largest subunit of RNA polymerase II and determine the factors that interact with this domain. While we are ultimately interested in human transcriptional regulation, we will also work extensively with yeast because of this superior genetics and its abundance and ability to substitute for human polymerase during transcription in vitro in extracts and purified systems. This work will build on our strengths, experience, and previous work on the identification, purification, and characterization of RNA polymerases and transcription factors. We will rely heavily on protein-protein and protein-DNA crosslinking; on the use of monoclonal antibodies to detect, inhibit, and immunopurify parts of the transcription apparatus; and on improved methods for efficient renaturation of proteins eluted from gels, blotted onto membranes, or from solubilized inclusion bodies. We are convinced that a basic, thorough study of RNA polymerase II structure and key interactions with transcription factors and DNA will provide major new insights into the mechanism of specific transcription and how that transcription is controlled. A long-term goal of this research is to use our detailed knowledge of specific polymerase- transcription factor interactions to design agents that interfere wit abnormal regulatory interactions crucial to maintaining the neoplastic state.