Structural studies of the eukaryotic transcription

描述(申请人提供)：基因转录的调控是 在细胞分化和发育过程中所必需的 生物体，以及每个细胞在不断变化的环境中的生存 条件。基因转录及其调控的重要性在于 突出的是由故障引起的无数人类疾病 参与这一细胞过程的分子成分。基因启动 真核生物中的转录始于TFIID的结合，包含 TATA结合蛋白(TBP)，到DNA核心启动子，以及由此而来的 通用转录机械的组装。六大通用转写 因子和RNA PolH构成了最小的蛋白质组装 转录，加起来有几百万道尔顿。TFIID还参与了 与基因特异性激活剂的相互作用。我们的最终目标是 了解真核转录启动机制是如何组装和 功能，以及基因转录调控的结构机制。至 为此，我们将描述TFIID的结构及其与 其他一般因素和活化剂使用冷冻电子显微镜和 单粒子图像重建。我们最近得到了一份初步的 TFHD及其与全长TFILA和TFIIB的35A络合物的结构 通过阴性染色分辨，并通过抗体映射定位位置 在建筑群内发现了TBP。我们的当务之急之一是获得一个3-D 以更好的分辨率重建冷冻水合的人TFIID复合体 超过25A。我们有兴趣遵循结构组装和 产生介质对这一复杂结构的构象变化 越来越大的络合物的分辨结构，以TFIID为 中央核心。我们的最终目标是描述 一般转录因子相互作用对转录调控的影响 带有激活器的机器。为此，我们将研究由 TFIID与通用激活剂Sp1。

DESCRIPTION (provided by applicant): Regulation of gene transcription is essential in the process of cell differentiation and development of the organism, as well as for the survival of each cell in changing environmental conditions. The importance of gene transcription and its regulation is underlined by the myriad of human disorders that result from the malfunction of the molecular components involved in this cellular process. Initiation of gene transcription in eukaryotes starts with the binding of TFIID, containing the TATA binding protein (TBP), to the DNA core promoter, and the consequent assembly of the general transcriptional machinery. Six general transcription factors and RNA Pol H constitute the minimal protein assembly for transcription, adding up to several million Daltons. TFIID is also involved in the interaction with gene-specific activators. Our ultimate goal is to understand how the eukaryotic transcription initiation machinery assembles and functions, and the structural mechanisms for gene transcription regulation. To this end we will characterize the structure of TFIID and its interaction with other general factors and with activators using cryo-electron microscopy and single-particle image reconstruction. We have recently obtained an initial structure of TFHD and its complex with full length TFILA and TFIIB at 35 A resolution by negative stain, and localized, by antibody mapping, the position of TBP within the complex. One of our immediate priorities is to obtain a 3-D reconstruction of frozen-hydrated human TFIID complexes at a resolution better than 25 A. We are interested in following the structural assembly and conformational changes of this complex machinery by generating medium resolution structures of increasingly larger complexes, with TFIID as the central core. Our final goal is to characterize the structural basis of transcription regulation by the interaction of the general transcriptional machinery with activators. To this end we will study the complexes formed by TFIID with the general activator Sp 1.