CRYSTALLOGRAPHY OF THE EUKARYOTE TRANSCRIPTION APPARATUS

真核生物转录装置的晶体学

• 批准号: 2388484
• 负责人: ROGER D KORNBERG
• 金额: $ 41.47万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-05-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2388484
• 关键词: DNA directed RNA polymerase; Saccharomyces cerevisiae; Schizosaccharomyces pombe; X ray crystallography; adenosinetriphosphatase; crystallization; cyclins; electron microscopy; enzyme complex; enzyme mechanism; enzyme structure; fungal genetics; genetic enhancer element; genetic promoter element; image processing; method development; nucleic acid sequence; protein structure function; transcription factor

The goal of our studies is the elucidation of RNA polymerase II structure, mechanism, and regulation. In previous work, methods were developed for 2-D crystallography of large multiprotein assemblies and applied to RNA polymerase II and its complexes with general transcription factors B and E. The results led to a hypothesis for the transcription initiation mechanism. We now propose the culmination of our efforts, with the structure determination of a complete 28-protein transcription initiation complex, and of an extended initiation complex including 16 additional proteins required for regulation. Structure determination of an actively transcribing complex will reveal the locations of template DNA and product RNA. Specific aims for the project period include the following: 1. Structure determination of RNA polymerase II in ice. 3-D reconstruction from electron micrographs of 2-D crystals is in progress. Extension of resolution by the use of single particle techniques and by combination of phase information from electron micrographs with intensities from X-ray diffraction will be undertaken. 2. Structure determination of the RNA polymerase II initiation complex. A series of RNA polymerase II-general transcription factor complexes will be solved by 2-D crystallography. The results will be combined to yield a picture of a complete RNA polymerase II initiation complex. 3. Structure determination of an RNA polymerase II transcription elongation complex 2-D crystals have been obtained of an actively transcribing complex, containing the polymerase paused at a single base on template DNA, with associated nascent RNA. 3-D reconstruction in ice is in progress. 4. Structure determination of general transcription factor H. a nine-subunit, 500,000 Da complex that possesses two ATPase/helicases and a cdk/cyclin pair, and which shares seven subunits with an even larger DNA repairosome. 5. Structure determination of a regulated RNA polymerase II initiation complex. We will undertake 2-D crystallography of the fifteen- subunit mediator, mediator-polymerase complex, and an extended polymerase II initiation complex, containing promoter DNA with an enhancer sequence, activator protein, mediator, polymerase, and general transcription factors.

我们研究的目标是阐明 RNA 聚合酶 II 结构、机制和规则。 在之前的工作中，方法 为大型多蛋白的二维晶体学而开发 组装并应用于 RNA 聚合酶 II 及其复合物 与一般转录因子 B 和 E 一起使用。结果导致 转录起始机制的假设。 我们现在 提出我们努力的顶峰，其结构 确定完整的 28 个蛋白质转录起始 复合物，以及扩展的起始复合物，包括 16 调节所需的额外蛋白质。 结构 主动转录复合物的测定将揭示 模板 DNA 和产物 RNA 的位置。具体目标为 项目周期包括以下内容： 一、结构 冰中RNA聚合酶II的测定。 3-D 重建 二维晶体电子显微照片的研究正在进行中。 通过使用单粒子技术扩展分辨率 通过结合电子显微照片的相位信息 将进行 X 射线衍射强度分析。 2. RNA 聚合酶 II 起始的结构测定 复杂的。 RNA聚合酶II系列-通用转录 因子复合物将通过二维晶体学来解析。 这 结果将被合并以产生完整 RNA 的图片 聚合酶 II 起始复合物。 3.结构测定 RNA 聚合酶 II 转录延伸复合物 2-D 已获得主动转录复合物的晶体， 含有在模板上的单个碱基处暂停的聚合酶 DNA，以及相关的新生RNA。 冰中的 3-D 重建是 进行中。 4. 一般转录的结构测定 因子 H. 一个包含 9 个亚基、500,000 Da 的复合物，具有两个 ATP酶/解旋酶和cdk/细胞周期蛋白对，它们共享七个 具有更大DNA修复体的亚基。 5. 结构 确定受调节的 RNA 聚合酶 II 起始 复杂的。 我们将进行十五种二维晶体学研究 亚基介体、介体聚合酶复合物和扩展 聚合酶 II 起始复合物，含有启动子 DNA 增强子序列、激活蛋白、介体、聚合酶、 和一般转录因子。