Mechanisms of eukaryotic transcription activation

真核转录激活机制

• 批准号: 7493054
• 负责人: Steven M Hahn
• 金额: $ 47.53万
• 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 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7493054
• 关键词: Binding; Biochemical; Biochemical Genetics; Biological Assay; C-terminal; Chromatin; Class; Complex; Development; Disease; Disruption; End Point; Gene Expression Regulation; Genes; Genetic Transcription; Hydroxyl Radical; In Vitro; Individual; Lead; Length; Malignant Neoplasms; Maps; Methods; Modeling; Molecular; Molecular Genetics; Mutagenesis; Mutation; N-terminal; Pathway interactions; Peptides; Proteins; RNA Polymerase II; Ribosomal Proteins; SAGA; Signal Transduction; Site; Staging; Structure; Tertiary Protein Structure; Testing; Transcription Coactivator; Transcriptional Activation; Transcriptional Regulation; Work; base; crosslink; in vivo; promoter; protein protein interaction; transcription factor

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 one of these targets, the SAGA coactivator complex, stimulates transcription. Our proposed work will illuminate important mechanisms and principles of transcriptional regulation.

转录激活是许多信号传输和发育途径的最终终点， 了解激活机制是了解这些途径作用的关键。 从之前的研究来看，很明显，基因特异性突变会破坏正常的基因调控。 转录激活剂可导致癌症和其他疾病。这一广泛的长期目标 建议确定基因特异性转录因子激活的机制 由 RNA 聚合酶 II (Pol II) 转录。拟议的工作将为理解基因提供基础 在分子水平上对正常和患病状态进行调节。 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检查 几种转录激活剂的直接靶标以及与这些靶标接触的机制 刺激转录。使用新开发的方法绘制大范围内的蛋白质-蛋白质接触图 复合物，我们将在存在或存在的情况下识别几种模型激活剂中激活域的直接目标 缺乏染色质。将进行生化和分子遗传学研究来测试相关性 转录中的这些相互作用。我们将扩展这些研究以检查不同激活剂的靶标 类在无 TATA 的发起人中起作用。通过羟基自由基绘制这些激活剂-靶标相互作用后 切割和直接蛋白质-蛋白质相互作用测定，我们将确定激活的结构 域与其相关目标相结合。最后，通过阻止预启动的组装 在中间阶段很复杂，我们将检查激活剂与其中之一接触的机制 这些目标，SAGA 共激活复合物，刺激转录。我们提出的工作将阐明 转录调控的重要机制和原理。