Mechanisms of eukaryotic transcription activation

真核转录激活机制

• 批准号: 7092225
• 负责人: Steven M Hahn
• 金额: $ 47.56万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7092225
• 关键词: DNA directed RNA polymerase; SDS polyacrylamide gel electrophoresis; Saccharomyces cerevisiae; animal tissue; antibody; chemical cleavage; chromatin; crosslink; enzyme structure; fungal genetics; genetic promoter element; genetic regulation; genetic transcription; hydroxyl radical; molecular genetics; molecular site; protein binding; protein protein interaction; ribosomal proteins; transcription factor

DESCRIPTION (provided by applicant): Activation of transcription is the ultimate endpoint for many signal transaction and developmental pathways, and understanding the mechanism of activation is a key to understanding the action of these pathways. From previous studies, it is clear that disruption of normal gene regulation by mutations in gene-specific transcription activators can lead to cancer and other diseases. The broad long-term objectives of this proposal are to determine the mechanisms used by gene-specific transcription factors to activate transcription by RNA Polymerase II (Pol II). 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 will utilize biochemical, structural, and molecular genetic methods to examine the direct targets of several transcription activators and the mechanism whereby contact with these targets stimulates transcription. Using a newly developed method for mapping protein-protein contacts within large complexes, we will identify direct targets of activation domains in several model activators in the presence or absence of chromatin. Biochemical and molecular genetic studies will be conducted to test the relevance of these interactions in transcription. We will extend these studies to examine the targets of a different activator class acting at TATA-less promoters. After mapping these activator-target interactions by hydroxyl radical cleavage and direct protein-protein interaction assays, we will determine the structure of the activation domains in combination with their relevant targets. Finally, by blocking assembly of the Preinitiation Complex at intermediate stages, we will examine the mechanism by which activator contact with 1 of these targets, the SAGA coactivator complex, stimulates transcription. Our proposed work will illuminate important mechanisms and principles of transcriptional regulation.

描述（申请人提供）：转录激活是许多信号传递和发育途径的最终终点，理解激活机制是理解这些途径作用的关键。从以前的研究中可以清楚地看出，基因特异性转录激活因子突变对正常基因调控的破坏可能导致癌症和其他疾病。这项提案的广泛的长期目标是确定基因特异性转录因子通过RNA聚合酶II（Pol II）激活转录的机制。这项工作将为在分子水平上理解正常和疾病状态下的基因调控提供基础。这项工作的具体目标将利用生物化学，结构和分子遗传学方法来研究几种转录激活因子的直接靶点，以及与这些靶点接触刺激转录的机制。使用一种新开发的方法映射蛋白质-蛋白质接触内的大型复合物，我们将确定直接目标的激活结构域在几个模型激活剂在染色质的存在或不存在。将进行生物化学和分子遗传学研究，以测试这些相互作用在转录中的相关性。我们将扩展这些研究，以检查在TATA-少启动子作用的不同激活剂类的目标。在通过羟基自由基裂解和直接蛋白质-蛋白质相互作用测定绘制这些激活剂-靶标相互作用之后，我们将确定激活结构域与其相关靶标的组合结构。最后，通过在中间阶段阻断预起始复合物的组装，我们将研究激活剂与这些靶点之一佐贺共激活剂复合物接触刺激转录的机制。我们的工作将阐明转录调控的重要机制和原则。