Mechanisms of eukaryotic transcription activation

描述(由申请人提供)： 摘要转录激活是基因调控的关键步骤，是控制细胞生长、发育和应激反应的许多信号通路的读出，而转录激活缺陷会导致许多人类疾病和综合征。激活通常是由含有激活结构域(ADS)的因子与含有激活剂结合结构域(ABD)的共激活复合体结合而产生的。大多数已知的广告在没有绑定合作伙伴的情况下是非结构化的，许多广告与多个结构无关的ABD交互。ADS与辅活化子相互作用的机制、这些相互作用的特异性以及辅活化子协同作用的机制是理解基因调控的分子基础的关键问题。该项目的长期目标是确定基因特异性激活子和辅助激活子用来调节RNA聚合酶(POL)II转录的机制。这项提案的目标是确定：ADS用来识别其共激活剂目标的机制，共激活剂Mediator和TFIID使用的合作机制，以及什么构成功能性AD和ABD。这项工作将利用生化、结构、分子和计算方法的跨学科组合来检查酿酒酵母的激活。这些机制很可能是保守的，因为几乎所有被测试的激活剂都在酵母和哺乳动物细胞中发挥作用。为了了解这些机制，我们将建立在过去赠款期间发展的几个突破性概念和方法的基础上。我们将使用蛋白质交联和质谱学，结合最先进的核磁共振来研究广泛作用的ADS在与其共激活靶结合时使用的机制-在大型生理相关复合体中和在原子水平上。我们将检查Mediator和TFIID之间的协作性，这两个保守的和广泛使用的转录辅助激活因子。最后，我们将使用分子、结构和计算方法的组合来描述AD功能的组成。这些新的方法将揭示激活剂结合和特异性的机制，协同激活剂合作的机制，并找到解释一大类广泛作用的广告的规则。我们的研究具有重要意义，因为它将揭示新的分子识别机制，这些机制对于理解转录调控以及涉及在许多生物系统中发挥功能的固有无序蛋白质的各种相互作用都是重要的。

DESCRIPTION (provided by applicant): Summary Transcription activation, a key step in gene control, is the readout of many signaling pathways controlling cell growth, development and stress response and defects in activation cause many human diseases and syndromes. Activation typically results from factors containing activation domains (ADs) binding to coactivator complexes containing activator binding domains (ABDs). Most known ADs are unstructured in the absence of a binding partner and many interact with multiple and structurally-unrelated ABDs. The mechanisms used by ADs to interact with coactivators, the specificity of these interactions, and mechanisms of coactivator cooperativity are critical unanswered questions for understanding the molecular basis of gene regulation. The long-term goal of this project is to determine mechanisms used by gene-specific activators and coactivators to regulate RNA polymerase (Pol) II transcription. The objectives of this proposal are to determine: mechanisms used by ADs to recognize their coactivator targets, cooperative mechanisms used by the coactivators Mediator and TFIID, and what constitutes a functional AD and ABD. This work will utilize an interdisciplinary combination of biochemical, structural, molecular, and computational approaches to examine activation in S. cerevisiae. These mechanisms are likely to be conserved, since nearly all activators tested function in both yeast and mammalian cells. To understand these mechanisms, we will build upon several breakthrough concepts and methods developed in the past grant period. We will use protein crosslinking and mass spectrometry, combined with state-of-the-art NMR to examine mechanisms used by broadly-acting ADs when binding their coactivator targets - in both large physiologically relevant complexes and at the atomic level. We will examine cooperativity between Mediator and TFIID, two conserved and widely used transcription coactivators. Finally, we will use a combination of molecular, structural, and computational approaches to delineate what comprises AD function. These new approaches will reveal mechanisms of activator binding and specificity, mechanisms of coactivator cooperation, and find rules that explain a large class of broadly acting ADs. Our proposed research is significant because it will reveal new molecular recognition mechanisms that are important for understanding transcriptional regulation as well as a wide variety of interactions involving inherently disordered proteins that function in many biological systems.