MOLECULAR MECHANISM OF TRANSCRIPTIONAL ACTIVATION

转录激活的分子机制

• 批准号: 2178102
• 负责人: William S. Dynan
• 金额: $ 22.11万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2178102
• 关键词: DNA; DNA directed RNA polymerase; DNA footprinting; RNA binding protein; adenosine triphosphate; affinity chromatography; autoantigens; chemical kinetics; chimeric proteins; enzyme activity; enzyme complex; enzyme mechanism; enzyme substrate; gel mobility shift assay; genetic promoter element; genetic transcription; human tissue; laboratory mouse; mass spectrometry; phosphorylation; protein kinase; protein structure function; site directed mutagenesis; stoichiometry; transcription factor; DNA; DNA directed RNA polymerase; DNA footprinting; RNA binding protein; adenosine triphosphate; affinity chromatography; autoantigens; chemical kinetics; chimeric proteins; enzyme activity; enzyme complex; enzyme mechanism; enzyme substrate; gel mobility shift assay; genetic promoter element; genetic transcription; human tissue; laboratory mouse; mass spectrometry; phosphorylation; protein kinase; protein structure function; site directed mutagenesis; stoichiometry; transcription factor

The long-term focus of this project is to understand the enzymatic mechanisms that underlie the response of RNA polymerase II to transcriptional activator proteins. These proteins regulate RNA synthesis in all cells and play a fundamental role in the control of cell growth and differentiation. Our recent efforts have concentrated on a template-associated protein kinase, DNA-PK, that phosphorylates the C-terminal domain of the largest subunit of RNA polymerase II at about the same time as RNA synthesis begins. We have purified the kinase to homogeneity and have identified its active components. Remarkably, the activity of the purified kinase is strongly stimulated by certain DNA-bound transcriptional activator proteins. This property suggests that the kinase may provide a means of transducing signals from transcriptional activators to the catalytic portion of the transcription apparatus. We propose experiments to better understand the mechanisms by which kinase activity is regulated, and to more clearly delineate the role of the kinase in transcription. Specific aims include: 1. Defining the functional role of kinase subunits. The kinase has a catalytic subunit and two regulatory subunits. We will study how the regulatory subunits affect kinase activity, and determine how the three subunits contact each other, the DNA, and the peptide substrate of the reaction. 2. Studying the interaction of the kinase with transcriptional activator proteins. We will determine how heat shock transcription factor and a fragment of yeast GAL4 stimulate kinase activity and will search for additional factors that may work by the kinase mechanism. 3. Determining the role of template-associated kinase in basal and activated transcription. We will test whether the presence of the kinase affects initiation, promoter clearance, or processivity. We will also test whether RNAP II phosphorylation functions as an energy-dependent proofreading step in initiation. 4. Developing novel probes of RNA polymerase II large subunit C-terminal domain (CTD) structure and function. We will map the contacts that the CTD makes with other transcription factors, generate novel anti-CTD reagents for use in vivo and in vitro, and identify functions of the CTD other than those related to phosphorylation.

该项目的长期重点是了解酶促 RNA聚合酶II响应的基础的机制 转录激活蛋白。这些蛋白质调节RNA合成 在所有细胞中，并在控制细胞生长和 分化。 我们最近的努力集中在模板相关蛋白上 激酶，DNA-PK，磷酸化最大的C末端结构域 RNA聚合酶II的亚基与RNA合成大约同时 开始。 我们已经将激酶纯化为均匀性，并确定了 其活性组件。值得注意的是，纯化激酶的活性是 由某些DNA结合的转录激活剂强烈刺激 蛋白质。该特性表明激酶可以提供 从转录激活剂到催化的转导信号 转录设备的一部分。 我们建议实验以更好 了解激酶活性受调节的机制，并 更清楚地描述了激酶在转录中的作用。 具体目的包括： 1。定义激酶亚基的功能作用。激酶有一个 催化亚基和两个调节亚基。我们将研究如何 调节亚基影响激酶活性，并确定三个 亚基相互接触，DNA和肽底物的接触 反应。 2。研究激酶与转录激活剂的相互作用 蛋白质。我们将确定热激转录因子和A 酵母gal4的片段刺激激酶活性，并将搜索 激酶机制可能起作用的其他因素。 3。确定模板相关激酶在基础和 激活的转录。我们将测试是否存在激酶 影响启动，启动子清除或加工性。我们还将测试 RNAP II磷酸化是否起到能量依赖性的作用 启动校对步骤。 4。开发RNA聚合酶II的新型探针II大亚基C末端 域（CTD）结构和功能。我们将绘制CTD的联系 与其他转录因子产生新型抗CTD试剂 用于体内和体外，并识别CTD以外的功能 与磷酸化有关的人。