Structure of RNA Polymerase II

描述(申请人提供)：拟议研究的目标是在原子分辨率下确定RNA聚合酶II及其与核酸和辅助蛋白质因子的复合体的X射线结构。这个问题很有挑战性，因为仅聚合酶就由15个多肽组成，总质量近60万道尔顿，加上辅助因子，多肽的数量和蛋白质质量都增加了一倍以上。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化和基因转录数据提供基础。下一个项目期的具体目标如下：1.揭示RNA聚合酶II转录复合体中RNA的退出路径。我们将确定一种含有34个残基转录本的新晶体的X射线结构。2.进行大型多组分转录延伸复合体的结构分析。我们将尝试结晶和解决RNA聚合酶II-Spt4/Spt5复合体的结构，作为确定整个P-TEFb/NELF/DSIF体系结构的一步，这具有特殊的临床意义和基础意义。3.阐明了RNA聚合酶II起始位点选择的机制。我们将继续研究关于通用转录因子TFIIB的“B指”结构域的结构及其在起始位置选择中的作用的最新发现。我们建议确定RNA聚合酶II与单独的B指、B指和一条模板DNA，以及B指、一条模板DNA和一个5个残基的RNA“转录本”的X射线结构。4.确定RNA聚合酶II-通用转录因子复合体的结构，最终目标是解决整个RNA聚合酶II的转录起始复合体。我们将尝试结晶和解决RNA聚合酶II与TFIIE、TFIIF、TFIIE和TFIIH的复合体，与TBP-TFIIB-启动子DNA复合体，与TBP-TFIIB-Tfg2ffFIIF-启动子DNA复合体，以及与所有组分的结构，包括处于闭合和开放状态的启动子DNA。

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.