Structure of the eukaryote transcription apparatus

这项资助支持了二维（2D）蛋白质晶体学的发展， 在25年的时间里，RNA的电子和X射线晶体结构 聚合酶II（pol II），单独和在转录的行为。这些结构 在上一个项目期间（2002-2007年）， 一般转录因子，阐明转录起始位点的机制 选择、启动子解链、失败启动和启动子逃逸。我们现在建议 我们努力的结果，整个转录机器的结构决定。 一般转录因子和转录调节因子的生物化学研究 转录调控在前一个项目期间已经删除了主要的 对拟议研究的障碍。初步的晶体学研究表明， 在下一个项目期间实现我们目标的可行性。 与2D晶体学和其他方法应用于 转录问题，我们追求的方法本身的发展。这 工作的灵感来自于一个想法，为例行的高分辨率结构测定大 复合物的电子显微镜和单颗粒分析。我们建议 将多个重原子簇刚性地连接到复合物上的特定位点， 成像，然后基于簇的投影位置对准图像。这 重原子电子显微镜（HevEM）方法需要完全均匀的团簇 直径约为一纳米，以及与生物结合的化学反应， 分子。在过去十年中，我们制定了满足这些要求的程序， 要求，并准备进行HevEM的首次试验，以及将其应用于 巨大的转录蛋白组装体 下一个项目期间的具体目标如下： 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。

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.