EUKARYOTIC TRANSCRIPTION INITIATION COMPLEX STRUCTURE

描述（由申请人提供）：基因转录是发育、肿瘤发生和细胞响应各种代谢和环境线索而不断重塑的基础：许多形态发生蛋白和癌蛋白是转录激活因子，转录是许多信号转导途径的终点。基础转录机制包括RNA聚合酶II（真核生物中负责合成所有信使RNA的酶）和五种辅助蛋白，称为一般转录因子。RNA聚合酶II和一般转录因子在启动子处组装形成前起始复合物，其包含超过30种不同的多肽并且具有几乎2 MDa的分子量。由于预引发复合物的尺寸和复杂性，常规技术如X射线晶体学和核磁共振光谱学无法提供其结构信息。本项目的总体目标是利用大分子电子显微镜和图像分析（理想地适合于大的，稀缺的大分子组装），以揭示酵母和人类preinitiation复合物在生理相关条件下的结构。一个逐步的方法将被应用：由RNA聚合酶和一般的转录因子形成的逐渐变大的复合物的结构将被确定，以下的preinitiation复合物在体内组装的顺序。RNA聚合酶在一般转录因子组装过程中所经历的结构变化以及整个前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。前起始复合物是介体的靶点，介体是一种多蛋白复合物，对于基础转录机制对来自激活子和阻遏子的调节信号的响应是必需的。我们将完成我们的工作，通过确定的结构的中介/preinitiation复杂的组件激活剂和阻遏物的存在。这些结构将指示调控信息被转导到前起始复合物的方式，并揭示真核细胞中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.