Mechanisms of eukaryotic transcription activation

描述（由申请人提供）： 转录激活是基因调控中的关键步骤，是控制细胞生长、发育和应激反应的许多信号通路的读出，并且激活的缺陷导致许多人类疾病和综合征。活化通常由含有活化结构域（AD）的因子与含有活化剂结合结构域（ABD）的共活化剂复合物结合引起。大多数已知的AD在不存在结合伴侣的情况下是非结构化的，并且许多与多个和结构上不相关的ABD相互作用。AD与辅激活因子相互作用的机制，这些相互作用的特异性，以及辅激活因子协同性的机制是理解基因调控的分子基础的关键未回答的问题。该项目的长期目标是确定基因特异性激活因子和共激活因子调节RNA聚合酶（Pol）II转录的机制。本提案的目标是确定：AD识别其辅激活因子靶点所使用的机制，辅激活因子Mediator和TFIID所使用的合作机制，以及功能性AD和ABD的构成。这项工作将利用生物化学，结构，分子和计算方法的跨学科组合来研究S。啤酒。这些机制可能是保守的，因为几乎所有的激活剂测试功能在酵母和哺乳动物细胞。为了理解这些机制，我们将建立在几个突破性的概念和方法，在过去的赠款期间开发。我们将使用蛋白质交联和质谱，结合最先进的NMR来研究广泛作用的AD在结合其共激活剂靶点时使用的机制-在大型生理相关复合物和原子水平上。我们将研究中介体和TFIID，两个保守的和广泛使用的转录辅激活因子之间的协同性。最后，我们将使用分子，结构和计算方法的组合来描绘什么构成AD功能。这些新方法将揭示激活剂结合和特异性的机制，辅激活剂合作的机制，并找到解释一大类广泛作用的AD的规则。我们提出的研究是重要的，因为它将揭示新的分子识别机制，这对于理解转录调控以及涉及在许多生物系统中起作用的固有无序蛋白质的各种相互作用非常重要。

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.