Structural Studies of the Eukaryotic Transcription Initiation Machinery

描述（由申请人提供）：转录的早期阶段对于真核生物中基因表达的调控至关重要。我们对这一过程的机械理解仍然非常有限，因为很难获得所涉及的分子参与者的大小和复杂性所带来的结构信息。我们正在使用电子显微镜和单粒子3-D重建来表征参与转录起始的大型人类转录复合物的结构、动力学和相互作用。在上一个资助期，我们确定了辅助因子CRSP和ARC-L、染色质重塑复合物PBAF以及一般因子TFIID和RNAPII的结构。对于最后两个复合物，我们的统计分析表明，在溶液中的构象之间的过渡，我们认为这反映了其功能所必需的机械阶段。我们将扩展这项工作，以表征RNAPII与非编码RNA（ncRNA）的抑制性相互作用，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？该信息如何在发起人处集成？这个建议的目标将阐明核心启动子识别的重要步骤，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。通过与完整的、功能性的人类复合物一起工作，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，一些相关晶体结构（例如酵母RNAPII、TBP、TFIIA. TFIIB）将为我们的重建提供附加值，通过将晶体学模型对接到我们的3-D密度中，使我们能够生成伪原子模型。最后，我们希望超越静态图片，能够利用我们的样品的溶液状态和新的图像重建方法和统计工具，描述这些动态复合物固有的构象景观，以及它们驱动的动态过程。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物的多方面过程。这些复合物中的大多数不能重组产生和重构，因此所有体外分析都需要从内源性物质中纯化。由于这些复合物的功能性质，它们在细胞中的存在量非常小，因此生物化学和生物物理学研究受到了极大的限制。这反过来又导致了几乎完全缺乏对这些复合物在分子水平上如何工作的机械理解。使用电子显微镜和单粒子图像分析，在大，低丰度复合物的结构表征的方法选择，我们提出了新的核心启动子识别的重要步骤，并在转录激活和转录抑制的过程中，在基因转录的早期阶段的行为。

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.