MOLECULAR MECHANISM OF TRANSCRIPTIONAL ACTIVATION

这个项目的长期重点是了解酶的 RNA聚合酶II的反应机制， 转录激活蛋白。这些蛋白质调节RNA合成 在所有细胞中，并在控制细胞生长中发挥重要作用， 分化 我们最近的努力集中在一个模板相关蛋白上 激酶DNA-PK，磷酸化最大的 RNA聚合酶II的亚基在大约与RNA合成同时 开始. 我们已经将激酶纯化至均一，并鉴定出 其活性成分。值得注意的是，纯化的激酶的活性是 受某些DNA结合转录激活因子的强烈刺激 proteins.这种性质表明，激酶可能提供了一种手段， 将信号从转录激活因子转导到催化的 转录器的一部分。 我们提出实验来更好地 了解激酶活性调节的机制，并 更清楚地描述了激酶在转录中的作用。 具体目标包括： 1.确定激酶亚基的功能作用。激酶具有 催化亚基和两个调节亚基。我们将研究 调节亚基影响激酶活性，并确定这三个 亚基彼此接触，DNA，和肽底物的 反应 2.研究激酶与转录激活因子的相互作用 proteins.我们将确定如何热休克转录因子和一个 酵母GAL 4片段刺激激酶活性，并将寻找 可能通过激酶机制起作用的其他因素。 3.确定模板相关激酶在基础和 激活转录。我们将测试激酶的存在是否 影响起始、启动子清除或持续合成能力。我们还将测试 RNAP II磷酸化是否作为一种能量依赖的 启动的校对步骤。 4. RNA聚合酶Ⅱ大亚基C端探针的研制 结构域（CTD）的结构和功能。我们会把反恐局 与其他转录因子结合，产生新型抗CTD试剂 用于体内和体外，并确定CTD的功能， 与磷酸化有关的。

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.