Structural Studies of the Eukaryotic Transcription Initiation Machinery

基因表达的转录调控是一项复杂的任务，对生长至关重要 和生存，无论是作为受精卵发育过程的一部分，还是 以适应不断变化的环境条件。转录起始步骤是 可以说是基因转录中最受调控的步骤，因为微调其速率和 同步可以作为一个关键控制点，产生生物体范围内基因变化， 表达谱响应发展和环境线索。 毫不奇怪，基因调控回路的复杂性是由大小决定的， 以及参与转录起始的分子的复杂性。过去 30年来，生物化学，分子遗传学和体内研究已经揭示了大多数，如果 而不是所有的转录机构的核心组成部分。但 对人类基因表达机制的理解是一个巨大的挑战 并且明显落后。一个主要的障碍是， 包括100多个单独的多肽， 由功能不同的多亚基复合物组成的组合，其中许多仅 可从内源获得。我们正在使用单粒子EM重建， 描述大型人类复合体的结构、动力学和相互作用 对基因调控至关重要。3D Cryo-EM重建是一种理想的技术， 这项任务，因为它需要有限的材料量，是最佳的研究非常大， 组装，并且具有检测和表征构象柔性的潜力， 一个性质，可以证明是至关重要的，能够描述所需的功能可塑性， 转录复合物，如TFIID，在这个过程中，需要结合 不同的DNA核心启动子，并整合来自各种各样的 转录激活因子和辅因子。 从根本上了解基因是如何影响人类健康的 转录是打开和关闭不能夸大。这种意义是 突出的事实是，各种癌症的发展伴随着 基因表达的改变导致疾病的各个方面， 诱导多能干细胞的发现，其中3-4个全球性的表达， 转录激活因子足以引入所需的基因表达变化， 转变为多能状态。通过了解蛋白质的结构， 基因激活，我们将指导未来的研究开发新的治疗方法， 其靶向调控介导细胞生长、瘤形成、转移 和血管生成，或那些旨在促进诱导的 多能干细胞的临床研究。

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, a property to 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.