Mechanisms of eukaryotic transcription activation

转录激活是许多信号转导和发育途径的最终终点，了解转录激活的机制是理解基因调控的关键。从以往的研究来看，基因特异性转录激活因子和辅激活因子突变对正常基因调控的破坏可导致癌症和其他疾病。本提案的长期目标是确定基因特异性激活因子和辅助激活因子调节RNA聚合酶II转录的机制。这项工作将为在分子水平上理解正常和患病状态下的基因调控提供基础。这项工作的具体目的是利用生化、结构和分子遗传学方法来检查两个激活域和两个共激活因子的直接靶点。我们将研究几种酸性激活剂-辅激活剂复合物的结构，以了解激活剂如何特异性识别它们的靶标，激活剂-靶标识别的共同原则，以及更一般地说，内在无序蛋白质的功能。我们将研究SAGA共激活子与TBP （TATA结合蛋白）的相互作用以及这种相互作用是如何通过乙酰化调节的。使用一套独特的交联试剂，我们将检查转录机制中的中介共激活子复合物的直接目标。在所有情况下，我们将使用酵母分子遗传学来测试我们的生化结果的功能意义。综上所述，我们的研究结果将为激活因子如何识别其协同激活因子靶标以及协同激活因子如何通过与转录机制的直接相互作用刺激基因表达提供一个分子模型。

DESCRIPTION (provided by applicant): Summary Activation of transcription is the ultimate endpoint for many signal transduction and developmental pathways, and understanding the mechanism of activation is a key to understanding gene regulation. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene- specific transcription activators and coactivators can lead to cancer and other diseases. The broad long- term objectives of this proposal are to determine the mechanisms used by gene-specific activators and coactivators to regulate RNA polymerase II transcription. The proposed work will provide a basis for understanding gene regulation in normal and diseased states at the molecular level. The specific aims of this work utilize biochemical, structural, and molecular genetic methods to examine the direct targets of two activation domains and two coactivators. We will examine the structure of several acidic activator-coactivator complexes to understand how activators specifically recognize their targets, common principals of activator-target recognition and, more generally, the function of intrinsically disordered proteins. We will examine the interaction of the SAGA coactivator with TBP (TATA binding protein) and how this interaction is regulated by acetylation. Using a unique set of crosslinking reagents, we will examine the direct targets of the Mediator coactivator complex within the transcription machinery. In all cases, we will use yeast molecular genetics to test the functional significance of our biochemical results. Combined, our results will lead to a molecular model for how activators recognize their coactivator targets and how coactivators stimulate gene expression by direct interaction with the transcription machinery. PUBLIC HEALTH RELEVANCE: Project Narrative The objective of this research is to understand the mechanism and regulation of transcription, the process of mRNA synthesis. Regulation of transcription is one of the key steps in control of cell growth, differentiation, and development, and defects in transcription directly contribute to many human illnesses. Understanding the mechanism of transcription and its regulation will form the basis for understanding the molecular defects in transcription disorders leading to many types of cancer, as well as heart disease, neurological disorders, and birth defects.