Structure of the eukaryote transcription apparatus

描述（由申请人提供）：该基金支持二维（2D）蛋白质晶体学的发展，在25年的时间里，导致了RNA聚合酶II（pol II）的电子和X射线晶体结构，单独和转录。在上一个项目期间（2002-2007年），这些结构被扩展到与一般转录因子的更高复合物，阐明了转录起始位点选择，启动子熔化，流产启动和启动子逃逸的机制。我们现在提出我们的努力的顶点，整个转录机制的结构确定。在上一个项目期间，对一般转录因子和转录调节介体的生物化学研究消除了拟议研究的主要障碍。初步的晶体学工作表明，在下一个项目期间实现我们的目标是可行的。在应用2D晶体学和其他方法来解决转录问题的同时，我们也在追求方法本身的发展。这项工作的灵感来自于一个想法，通过电子显微镜和单粒子分析的常规高分辨率结构测定的大型复合物。我们提出了在成像之前将多个重原子团簇刚性地附着到复合物上的特定位点，然后基于团簇的投影位置对齐图像。这种重原子电子显微镜（HevEM）方法需要直径约为纳米的完全均匀的簇，以及与生物分子结合的化学反应。在过去的十年中，我们已经设计出满足这些要求的程序，并准备进行HevEM的首次试验，以及将其应用于感兴趣的巨大转录蛋白组装体。下一个项目期的具体目标如下：1.通用转录因子H的结构测定。2. pol Ⅱ-通用转录因子复合物的结构测定。3.介体和介体- pol II复合物的结构测定。4.开发HevEM方法。公共卫生相关性：这项研究的意义可以概括为：它将为全面理解转录的基本机制提供所需的结构信息;它将为转录调控的研究建立结构基础;它将促进未来异常基因调控疾病的新疗法的设计。

DESCRIPTION (provided by applicant): This grant supported the development of two-dimensional (2D) protein crystallography, which led, over the course of 25 years, to electron and X-ray crystal structures of RNA polymerase II (pol II), alone and in the act of transcription. These structures were extended, during the previous project period (2002-2007), to higher complexes with general transcription factors, illuminating the mechanisms of transcription start site selection, promoter melting, abortive initiation, and promoter escape. We now propose the culmination of our efforts, the structure determination of the entire transcription machinery. Biochemical studies of the general transcription factors and of the Mediator of transcriptional regulation during the previous project period have removed the major obstacles to the proposed research. Preliminary crystallographic work has shown the feasibility of accomplishing our goal during the next project period. In parallel with the application of 2D crystallography and other methods to the transcription problem, we have pursued the development of the methodology itself. This work was inspired by an idea for routine high resolution structure determination of large complexes by electron microscopy and single particle analysis. We have proposed to rigidly attach multiple heavy atom clusters to specific sites on the complexes before imaging, and then align the images based on the projected positions of the clusters. This heavy atom electron microscopy, or "HevEM," approach requires perfectly uniform clusters about a nanometer in diameter, as well as chemistries for their conjugation with biological molecules. During the past decade, we have devised procedures for meeting these requirements, and are poised for the first trial of HevEM, as well as its application to the giant transcription protein assemblies of interest. Specific aims for the next project period are as follows: 1. Structure determination of general transcription factor H. 2. Structure determination of pol II - general transcription factor complexes. 3. Structure determination of Mediator and of Mediator - pol II complexes. 4. Development of the HevEM approach. PUBLIC HEALTH RELEVANCE: The significance of the proposed research may be summarized as follows: it will provide the structural information needed to fully understand the fundamental mechanism of transcription; it will establish a structural basis for studies of transcriptional regulation; and it will facilitate the design of new therapies for diseases of aberrant gene regulation in the future.