MOLECULAR MECHANISM OF TRANSCRIPTIONAL ACTIVATION

这个项目的长期重点是了解酶 RNA聚合酶II应答的机制 转录激活蛋白。这些蛋白质调节rna的合成。 在所有细胞中，并在控制细胞生长和 差异化。 我们最近的努力集中在一种模板相关蛋白质上 激酶，DNA-PK，磷酸化最大的 与RNA合成几乎同时的RNA聚合酶II亚基 开始。我们已经将该激酶提纯为均一，并鉴定出 它的活性成分。值得注意的是，纯化的激酶的活性是 某些DNA结合转录激活剂的强烈刺激 蛋白质。这一特性表明，该激酶可能提供了一种 从转录激活子到催化剂的信号转导 转录装置的一部分。我们建议进行实验，以更好地 了解调节激酶活性的机制，并 更清楚地描述了该激酶在转录中的作用。 具体目标包括： 1.明确了激酶亚基的功能作用。该激酶有一种 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，并决定三个亚基如何 亚基之间相互联系，DNA和多肽底物 反应。 2.研究蛋白激酶与转录激活剂的相互作用 蛋白质。我们将确定热休克转录因子和一个 酵母GAL4片段刺激激酶活性并将寻找 可能通过激酶机制发挥作用的其他因素。 3.确定模板相关激酶在基础和非基础状态中的作用 激活了转录。我们将测试该激酶的存在是否 影响启动、启动子清除或过程。我们还将测试 RNAP II磷酸化是否作为一种能量依赖的 启动中的校对步骤。 4.RNA聚合酶II大亚基C末端新型探针的研制 域(CTD)结构和功能。我们会将CTD的联系人 与其他转录因子共同作用，产生新型抗CTD试剂 在体内和体外使用，并鉴定CTD的功能 那些与磷酸化有关的基因。

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.