EUKARYOTIC TRANSCRIPTION INITIATION COMPLEX STRUCTURE

描述(申请人提供)：基因转录是细胞发育、肿瘤发生和对各种代谢和环境信号作出反应的持续重塑的基础：许多形态原和癌蛋白是转录激活剂，而转录是许多信号转导途径的终点。基本的转录机制包括RNA聚合酶II(负责合成真核生物中所有信使RNA的酶)和五种辅助蛋白，称为通用转录因子。RNA聚合酶II和一般转录因子聚集在启动子上形成一个预起始复合体，该复合体由30多个不同的多肽组成，分子质量接近2丙二醛。预引发复合体的大小和复杂性使X射线结晶学和核磁共振光谱等传统技术无法提供有关它的结构信息。这个项目的总体目标是使用大分子电子显微镜和图像分析(非常适合于大的、稀缺的大分子组件的结构表征的技术)来揭示与生理相关的条件下酵母和人类预引发复合体的结构。将采用循序渐进的方法：按照预起始复合体在体内的组装顺序，确定由RNA聚合酶和一般转录因子形成的逐渐变大的复合体的结构。RNA聚合酶在一般转录因子组装时所经历的结构变化，以及整个预起始复合体的拓扑结构，将揭示转录起始和启动子逃逸的机制。预起始复合体是Mediator的靶标，Mediator是一种多蛋白复合体，对于基础转录机制对来自激活物和抑制物的调节信号的反应是必不可少的。我们将在激活剂和阻滞剂存在的情况下，通过确定介体/预引发复合体组件的结构来完成我们的工作。这些结构将指示调控信息传递到预起始复合体的方式，并揭示真核细胞转录调控机制的结构基础。

DESCRIPTION (provided by applicant): Gene transcription underlies development, oncogenesis, and the constant reshaping of the cell in response to a variety of metabolic and environmental cues: many morphogens and oncoproteins are transcriptional activators, and transcription is the endpoint of a number of signal transduction pathways. The basal transcription machinery comprises RNA polymerase II (the enzyme responsible for synthesis of all messenger RNA in eukaryotes) and five accessory proteins, known as general transcription factors. RNA polymerase II and the general transcription factors assemble at a promoter to form a preinitiation complex, which comprises over 30 different polypeptides and has a molecular mass of almost 2 MDa. The size and complexity of the preinitiation complex have prevented conventional techniques such as X-ray crystallography and NMR spectroscopy from providing structural information about it. The overall goal of this project is to employ macromolecular electron microscopy and image analysis (techniques that are ideally suited for the structural characterization of large, scarce macromolecular assemblies) to reveal the structure of the yeast and human preinitiation complexes under physiologically-relevant conditions. A step-wise approach will be applied: structures of progressively larger complexes formed by RNA polymerase and the general transcription factors will be determined, following the order of assembly of the preinitiation complex in vivo. The structural changes that RNA polymerase undergoes as the general transcription factors assemble, and the topology of the entire preinitiation complex, will reveal the mechanism of transcription initiation and promoter escape. The preinitiation complex is the target of Mediator, a multiprotein complex that is essential for response of the basal transcription machinery to regulatory signals from activators and repressors. We will complete our work by determining the structure of the Mediator/preinitiation complex assemblies in the presence of activators and repressors. The structures will indicate the way in which regulatory information is transduced to the preinitiation complex, and reveal the structural underpinnings of the transcription regulation mechanism in eukaryotic cells.