RNA POLYMERASE II AND THE TRANSCRIPTION COMPLEX

在转录水平上调节基因表达是 调节细胞生长和分化的主要手段。异常 转录调控在肿瘤中发挥重要作用 转型。尽管在这方面有大量的研究活动 转录调控机制，我们才刚刚开始了解 多个转录因子及其如何促进的图片， 激活并抑制特定的转录。目前很少有 了解RNA聚合酶中亚基相互作用的细节 II这些亚单位如何与细胞相互作用并受其调节 大量的转录因子。我们将集中精力在结构上， 转录机制的功能和调节，重点是 关于RNA聚合酶II。我们将研究RNA聚合酶II亚单位 组织：通过蛋白质-蛋白质鉴定稳定的亚组分 并通过合作确定其3-D结构 酵母酶。我们将确定RNA聚合酶II的哪些亚基是 参与与一般转录因子的相互作用 溶液和再引发复合体中的因子-聚合酶复合体 在最小启动子系统上形成(涉及人免疫球蛋白启动子， RNA聚合酶II与最基本的通用转录因子 TBP、TFIIB和RAP30)。我们将推广我们最近的研究成果 RNA最大亚基的C-末端结构域的重要性 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， 由于这一优势，我们还将广泛使用酵母 遗传学及其替代人类聚合酶的丰度和能力 在提取物和纯化系统中的体外转录过程中。这 我们的工作将建立在我们的优势、经验和之前关于 RNA聚合酶的鉴定、纯化和性质研究 转录因子。我们将严重依赖蛋白质-蛋白质和 蛋白质-DNA交联；关于使用单克隆抗体来检测， 抑制和免疫纯化部分转录装置；以及 从凝胶中洗脱的蛋白质高效复性的改进方法， 印迹到膜上，或从溶解的包涵体中。 我们相信，对RNA聚合酶II的基本、彻底的研究 转录因子和DNA Will的结构和关键相互作用 提供了对特定转录机制的重大新见解 以及转录是如何被控制的。这是一个长期目标 研究是利用我们对特定聚合酶的详细知识- 转录因子相互作用以设计干扰WITS的试剂 异常的调节相互作用对维持肿瘤至关重要 州政府。

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.