Structural Studies of the Eukaryotic Transcription Initiation Machinery

描述（由申请人提供）：基因表达的转录调控是一项复杂的任务，对生长和生存至关重要，无论是作为受精卵发育过程的一部分，还是在适应不断变化的环境条件时。转录起始步骤可以说是基因转录中最受调控的步骤，因为对其速率和同步性的微调可以作为一个关键控制点，以响应发育和环境线索，在整个生物体范围内产生基因表达谱的变化。毫不奇怪，基因调控回路的复杂性与参与转录起始的分子参与者的大小和复杂性是平行的。在过去的30年里，生物化学、分子遗传学和体内研究已经揭示了大部分（如果不是全部的话）转录装置的核心组成部分。然而，对人类基因表达的机械理解是一个巨大的挑战，并且远远落后。一个主要的障碍是转录机制包括超过100个单独的多肽，这些多肽作为一个巨大的、动态的组合，由功能不同的多亚基复合物组成，其中许多只能从内源性来源获得。我们正在使用单粒子EM重建来表征基因调控所必需的大型人类复合物的结构，动力学和相互作用。3D冷冻电镜重建是一种非常适合这项任务的技术，因为它需要的材料数量有限，最适合研究非常大的组件，并且具有检测和表征构象灵活性的潜力。后者的特性对于描述转录复合体（如TFIID）所需的功能可塑性至关重要，TFIID是该过程中的重要枢纽，需要结合不同的DNA核心启动子并整合来自各种转录激活子和辅因子的输入。对基因转录如何开启和关闭的基本理解对人类健康的重要性怎么强调都不为过。各种癌症的发展都伴随着基因表达的改变，导致疾病的各个方面，以及诱导多能干细胞的发现，其中3-4个全局转录激活因子的表达足以引入过渡到多能状态所需的基因表达变化，这一事实凸显了这种意义。通过了解基因激活所需的蛋白质结构，我们将指导未来的研究，以控制基因表达为目标，介导人类细胞生长、肿瘤、转移和血管生成，或旨在促进诱导多能干细胞研究向临床的转变。

DESCRIPTION (provided by applicant): Transcriptional regulation of gene expression is a complex task, critical for growth and survival, whether as part of the developmental process from the fertilized egg, or when adapting to changing environmental conditions. The transcription initiation step is arguably the most regulated step in gene transcription, as fine tuning both its rate and synchrony can serve as a key control point to produce organism-wide changes in gene expression profiles in response to developmental and environmental cues. Not surprisingly, the complexity of gene regulatory circuitries is paralleled by the size and complexity of the molecular players involved in transcription initiation. Over the past 30 years, biochemistry, molecular genetics, and in vivo studies have uncovered most, if not all, of the central components of the transcriptional apparatus. However, a mechanistic understanding of gene expression in humans poses a formidable challenge and lags dramatically behind. A major obstacle is that the transcriptional machinery comprises more than 100 individual polypeptides that operate as a huge and dynamic assemblage made up of functionally distinct multi-subunit complexes, many of them only accessible from endogenous sources. We are using single particle EM reconstruction to characterize the architecture, dynamics and interactions of large human complexes essential for gene regulation. 3D Cryo-EM reconstruction is a technique ideally suited to this task, as it requires limited amounts of material, is optimal to study very large assemblies, and is has the potential to detect and characterize conformational flexibility. The latter is a property that may prove critical to be able to describe the functional plasticity required in transcriptional complexes like TFIID, an essential hub in this process that needs to bind to different DNA core promoters and integrate the input from a large variety of transcriptional activators and cofactors. The significance to human health of a fundamental understanding of how gene transcription is switched on and off cannot be overstated. Such significance is highlighted by the fact that development of various cancers is accompanied by alterations in gene expression leading to various aspects of the disease, and by the discovery of induced pluripotent stem cells, where the expression of 3-4 global transcriptional activators is sufficient to introduce gene expression changes needed to transition into a pluripotent state. By understanding the protein structures necessary for gene activation, we will guide future research into the development of novel treatments that target the control of gene expression mediating cell growth, neoplasia, metastasis, and angiogenesis in humans or those aimed at facilitating the transition of induced pluripotent stem cells research into the clinic.