CRYSTALLOGRAPHY OF THE EUKARYOTE TRANSCRIPTION APPARATUS

我们的长期目标是了解生物多样性的机制和调节。 真核生物中的转录。下一个项目期的目标是 确定由一组由一组 五种通用转录因子和RNA聚合酶II在一个启动子。 这项工作将涉及分子遗传学、生化、电子和X射线。 结晶学研究以及与之相关的技术发展 电子结晶学方法。研究的具体目的如下 以下是： 1.绘制二维晶体中RNA聚合酶II的表面形貌图 16埃分辨率。11个亚基中的8个亚基的位置 酶，包括最大亚基的C-末端重复结构域， 在监管中的重要性，将被确定。与之互动的站点 一般转录因子将被确定。DNA和RNA结合 各地区将被划定。 2.高分辨率测定RNA聚合酶II的结构。一个 X-射线晶体分析与电子显微镜相结合 2-D，将使用晶体。酶-DNA复合体的结构和 转录延伸复合体，包含酶、DNA和RNA将 也被调查。 3.测定一般转录因子的高水平结构 决议。TFIID的结构将从以下晶体中衍生 衍射到2.5埃分辨率，以及四个额外的一般 这些因素也将接受结晶学分析。 4.确定转录因子的排列，RNA聚合酶 和启动子DNA在起始复合体中的电子显微镜和 二维晶体的图像处理。结果将与那些 从X射线结晶学分析得出的各个成分 整个建筑群的高分辨率结构。 5.继续发展脂层结晶法 生长二维蛋白质晶体。方法和解决方案的范围 衍生的结构信息将通过工程小说进行扩展 通过改善蛋白质与脂层结合的适配器， 2-D晶体的保存，并通过对 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.