CRYSTALLOGRAPHY OF THE EUKARYOTE TRANSCRIPTION APPARATUS

我们的长期目标是了解 真核生物中的转录。 下一个项目期间的目标是 以确定由一组 五个通用转录因子和启动子处的RNA聚合酶II。 这项工作将涉及分子遗传学、生物化学、电子和X射线 晶体学研究，以及相关的技术发展， 电子晶体学方法 研究的具体目标是 如下所示： 1. 为了在二维晶体中绘制RNA聚合酶II的表面形貌， 16埃分辨率。 11个亚单位中的8个的位置 酶，包括最大亚基的C末端重复结构域， 在监管方面，这将是决定性的。 相互作用部位 将鉴定通用转录因子。 DNA和RNA结合 将划定区域。 2. 以高分辨率确定RNA聚合酶II的结构。 一 结合X射线晶体学分析和电子显微镜， 2-D，将采用晶体。 酶-DNA复合物的结构和 含有酶、DNA和RNA的转录延伸复合物将 也被调查。 3. 为了确定一般转录因子的结构， 分辨率 TFIID的结构将来自晶体， 分辨率为2.5埃，另外四个一般 也将对因子进行晶体学分析。 4. 为了确定转录因子的排列，RNA聚合酶 II，和启动子DNA的起始复合物，通过电子显微镜和 二维晶体的图像处理。 结果将与那些 通过对单个成分的X射线晶体学分析 整个建筑群的高分辨率结构。 5. 继续开发脂质层结晶方法， 生长二维蛋白质晶体 方法和分辨率的范围 所得到的结构信息将被工程新颖性所扩展 用于将蛋白质结合到脂质层的衔接子， 保存2-D晶体，并进行基础研究， 二维结晶过程。

Our long range goal is to understand the mechanism and regulation of transcription in eukaryotes. The objective for the next project period is to determine the structure of the initiation complex formed by a set of five general transcription factors and RNA polymerase II at a promoter. The work will entail molecular genetic, biochemical, electron and X-ray crystallographic studies, as well as technical development related to the electron crystallographic approach. Specific aims of the research are as follows: 1. To map the surface topography of RNA polymerase II in 2-D crystals at 16 angstrom resolution. The locations of eight of eleven subunits of the enzyme, including the C-terminal repeat domain of the largest subunit, important in regulation, will be determined. Sites of interaction with general transcription factors will be identified. DNA and RNA-binding regions will be delineated. 2. To determine the structure of RNA polymerase II at high resolution. A combination of X-ray crystallographic analysis and electron microscopy of 2-D, crystals will be employed. The structures of enzyme-DNA complexes and transcription elongation complexes, containing enzyme, DNA, and RNA will also be investigated. 3. To determine the structures of general transcription factors at high resolution. The structure of TFIID will be derived from crystals that diffract to 2.5 angstrom resolution, and the four additional general factors will be subjected to crystallographic analysis as well. 4. To determine the arrangement of transcription factors, RNA polymerase II, and promoter DNA in the initiation complex by electron microscopy and image processing of 2-D crystals. The results will be combined with those from X-ray crystallographic analysis of the individual components to arrive at a high resolution structure of the entire complex. 5. To continue development of the lipid layer crystallization approach for growing 2-D protein crystals. The range of the approach and resolution of structural information derived will be extended by engineering novel adaptors for binding proteins to lipid layers, by improving the preservation of 2-D crystals, and by conducting fundamental studies of the 2-D crystallization process.