Structural Studies of the Eukaryotic Transcription Initiation Machinery

描述(申请人提供)：转录的早期阶段对真核生物的基因表达调控至关重要。我们对这一过程的机械理解仍然非常有限，因为所涉及的分子参与者的大小和复杂性带来了获得结构信息的困难。我们正在使用电子显微镜和单粒子三维重建来表征参与转录起始的大型人类转录复合体的结构、动力学和相互作用。在之前的资助期间，我们确定了辅助因子CRSP和ARC-L的结构，染色质重塑复合体PBAF，以及通用因子TFIID和RNAPII。对于后两个络合物，我们的统计分析表明，溶液中构象之间的转变，我们认为这反映了对它们的功能至关重要的机制阶段。我们将继续这项工作，以表征RNAPII与非编码RNA(NcRNAs)的抑制相互作用，并研究TFIID与核心启动子DNA、TFIIA和TFIIB以及人类介体的结合。绑定发生在哪里？结合对构象和动力学有什么影响？这些信息是如何在发起人处整合的？这项提议的目标将阐明核心启动子识别的基本步骤，以及在基因转录的非常早期阶段起作用的转录激活和转录抑制过程。通过使用完整的、具有功能的人类复合体，我们的工作将更好地反映在细胞环境中发生的蛋白质-蛋白质和蛋白质-核酸相互作用的程度和复杂性。另一方面，一些相关的晶体结构(例如酵母RNAPII，TBP，TFIIA.，TFIIB)的出现将为我们的重建带来额外的价值，因为它允许我们通过将晶体模型与我们的3-D密度对接来生成伪原子模型。最后，我们希望超越静态图片，通过能够开发我们的样本的溶液状态和新的图像重建方法和统计工具，来描述这些动态复合体固有的构象景观，以及它们所驱动的动态过程。公共卫生相关性：真核生物的转录调控是一个涉及大量蛋白质复合体的多方面过程。这些复合体中的大多数不能重组生产和重组，因此所有的体外分析都需要从内源性物质中提纯。鉴于这些复合体由于其功能的性质，在细胞中以非常少量的形式存在，生物化学和生物物理研究受到了极大的限制。这反过来又导致几乎完全缺乏对这些络合物在分子水平上如何工作的机制的了解。利用电子显微镜和单粒子图像分析方法，我们建议对核心启动子识别的基本步骤以及在基因转录的非常早期阶段起作用的转录激活和转录抑制过程提供新的线索。

DESCRIPTION (provided by applicant): The early stages of transcription are critical for regulation of gene expression in eukaryotes. Our mechanistic understanding of this process remains very limited due to the difficulty in obtaining structural information brought about by the size and complexity of the molecular players involved. We are using electron microscopy and single particle 3-D reconstruction to characterize the architecture, dynamics and interactions of large human transcription complexes involved in transcription initiation. In the previous funding period we determined the architecture of co-factors CRSP and ARC-L, the chromatin remodeling complex PBAF, and the general factors TFIID and RNAPII. For the last two complexes, our statistical analysis showed that the transition between conformations in solution, which we believe reflect mechanistic stages essential to their function. We will extend this work to characterize the repressive interaction of RNAPII with non-coding RNAs (ncRNAs), and to study the binding of TFIID to core promoter DNA, TFIIA and TFIIB, and human Mediator. Where does binding occur? What is the effect of binding on conformation and dynamics? How is this information integrated at the promoter? The goals of this proposal will shed light on the essential step of core promoter recognition, and on processes of transcriptional activation and transcriptional repression that act at the very early stages of gene transcription. By working with full, functional human complexes, our work will better reflect the extent and complexity of protein-protein and protein-nucleic acid interactions that occur in the cellular context. On the other hand, the availability of some relevant crystallographic structures (e.g. yeast RNAPII, TBP, TFIIA., TFIIB) will give added value to our reconstructions, by allowing us to generate pseudo-atomic models via docking of the crystallographic models into our 3-D densities. Finally, we hope to go beyond static pictures, by being able to exploit the solution state of our samples and new image reconstruction methods and statistical tools, to describe the conformational landscape intrinsic to these dynamic complexes, and to the dynamic processes they drive. PUBLIC HEALTH RELEVANCE: Transcriptional regulation in eukaryotes is a multifaceted process involving a very large number of protein complexes. Most of these complexes cannot be produced recombinantly and reconstituted, so that all in vitro analysis requires purification from endogenous material. Given that these complexes, by nature of their function, exist in very small amounts in the cell, biochemical and biophysical studies have been significantly limited. This in turn has lead to an almost complete lack of mechanistic understanding of how these complexes work at the molecular level. Using electron microscopy and single particle image analysis, the methodology of choice in the structural characterization of large, low-abundance complexes, we propose to shed new light on the essential step of core promoter recognition, and on processes of transcriptional activation and transcriptional repression that act at the very early stages of gene transcription.