Structure of RNA Polymerase II

描述（由申请人提供）：拟议研究的目标是以原子分辨率确定RNA聚合酶II及其与核酸和辅助蛋白因子的复合物的X射线结构。该问题是具有挑战性的，因为单独的聚合酶包含15种多肽，总质量接近600，000道尔顿，并且添加辅助因子使多肽的数量和蛋白质质量加倍以上。拟议的研究代表了我们努力的顶峰，并将为理解过去30年来在不同系统中积累的大量生物化学和遗传数据提供基础。下一个项目期的具体目标如下：1.揭示了RNA聚合酶II转录复合体中RNA的出口路径。我们将确定一种含有34个残基的转录物的新晶体的X射线结构。2.进行大型多组分转录延伸复合物的结构分析。我们将尝试结晶和解决RNA聚合酶II -Spt 4/Spt 5复合物的结构，作为整个P-TEFb/NELF/DSIF系统的结构确定的一个步骤，这具有特殊的临床意义，以及基础意义。3.阐明了RNA聚合酶II的起始位点选择机制。我们将追求最近的研究结果有关的“B指”结构域的一般转录因子TFIIB和它的作用，在起始位点的选择。我们建议确定RNA聚合酶II复合物的X射线结构，包括单独的B指、B指和一条模板DNA链以及B指、一条模板DNA链和一个5残基RNA“转录物。“4.确定RNA聚合酶II -通用转录因子复合物的结构，最终目标是解决整个RNA聚合酶II转录起始复合物。我们将尝试结晶和解析RNA聚合酶II与TFIIE、TFIIF、TFIIE和TFIIH、TBP - TFIIB -启动子DNA复合物、TBP-TFIIB -Tfg 2ffFIIF-启动子DNA复合物以及所有组分（包括处于“闭合”和“开放”状态的启动子DNA）的复合物的结构.

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the X-ray structure of RNA polymerase II and of its complexes with nucleic acids and auxiliary protein factors at atomic resolution. The problem is challenging, since the polymerase alone comprises 15 polypeptides with a total mass of nearly 600,000 Daltons, and addition of the auxiliary factors more than doubles both the number of polypeptides and the protein mass. The proposed research represents the culmination of our efforts, and will provide a basis for understanding a vast body of biochemical and genetic data on transcription accumulated in diverse systems over the past 30 years. Specific aims for the next project period are as follows: 1. Reveal the exit path of RNA from an RNA polymerase II transcribing complex. We will determine the X-ray structure of a new crystal containing a 34-residue transcript. 2. Undertake the structural analysis of large, multicomponent transcription elongation complexes. We will attempt to crystallize and solve the structure of RNA polymerase II - Spt4/Spt5 complexes, as a step towards the structure determination of the entire P-TEFb/NELF/DSIF system, which has particular clinical, as well as fundamental significance. 3. Elucidate the mechanism of RNA polymerase II start site selection. We will pursue recent findings concerning the structure of the "B finger" domain of general transcription factor TFIIB and its role in start site selection. We propose to determine the X-ray structures of RNA polymerase II complexes with the B finger alone, with the B finger and a strand of template DNA, and with the B finger, a strand of template DNA, and a 5-residue RNA "transcript." 4. Determine the structures of RNA polymerase II - general transcription factor complexes, with the ultimate goal of solving the entire RNA polymerase II transcription initiation complex. We will attempt to crystallize and solve the structures of RNA polymerase II complexes with TFIIE, with TFIIF, with TFIIE and TFIIH, with a TBP - TFIIB - promoter DNA complex, with TBP - TFIIB - Tfg2ffFIIF - promoter DNA complexes, and with all components, including promoter DNA in both "closed" and "open" states.