Structure of the eukaryote transcription apparatus

描述(申请人提供)：这笔赠款支持二维(2D)蛋白质结晶学的发展，在25年的时间里，它单独和在转录行为中导致了RNA聚合酶II(PolII)的电子和X射线晶体结构。在之前的项目期间(2002-2007)，这些结构被延伸到与一般转录因子的高级复合体，阐明了转录起始点选择、启动子熔化、流产起始和启动子逃逸的机制。我们现在提出我们努力的顶峰，整个转录机器的结构确定。在之前的项目期间，对一般转录因子和转录调控中介的生物化学研究已经消除了拟议研究的主要障碍。初步的结晶学工作表明，在下一个项目期间实现我们的目标是可行的。在将2D结晶学和其他方法应用于转录问题的同时，我们一直在追求方法学本身的发展。这项工作的灵感来自于通过电子显微镜和单粒子分析来确定大分子络合物的常规高分辨率结构的想法。我们提出了在成像前将多个重原子团簇刚性连接到络合物上的特定位置，然后根据团簇的投影位置对齐图像。这种重原子电子显微镜或“HevEM”方法需要直径约一纳米的完美均匀的团簇，以及它们与生物分子结合的化学成分。在过去的十年中，我们设计了满足这些要求的程序，并准备进行HevEM的第一次试验，以及将其应用于感兴趣的巨型转录蛋白组件。下一个项目期的具体目标如下：1.通用转录因子的结构测定H.2.PolII-通用转录因子复合体的结构测定。3.介体和介体-PolII络合物的结构测定。4.HevEM方法的发展。与公共健康相关：拟议研究的意义可以概括如下：它将提供充分理解转录基本机制所需的结构信息；它将为转录调控研究奠定结构基础；它将促进未来针对基因调控异常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.