Functional Characterization of Nuclear Myosin I in Transcription

描述(申请人提供)：核肌球蛋白I是肌球蛋白超家族肌球蛋白激活的ATPase的成员，发现于细胞核。也有大量证据表明细胞核中存在肌动蛋白。核肌球蛋白I和肌动蛋白是RNA聚合酶I和II转录所必需的。具体地说，肌动蛋白是RNA聚合酶II形成预起始复合体所必需的，肌动蛋白与RNA聚合酶I、II和III结合。我们还发现核肌球蛋白I参与转录起始，肌动蛋白和核肌球蛋白I是延长复合体的组成部分。基于大量的初步数据，我们提出了核肌球蛋白I在RNA聚合酶II的转录延伸中起关键作用的假说。具体目标1有两个部分。在第一部分，我们将分析固定在固定DNA模板上的延长的复合体，以确定核肌球蛋白I的结合伙伴。在第二部分，我们将重新启动暂停的复合体，以确定核肌球蛋白I在延长中的作用。特异性目标2将直接研究肌动蛋白和核肌球蛋白I在转录延伸中具有运动功能的可能性。由于RNA聚合酶II也是水解三磷酸腺苷的马达，我们重新设计了DNA模板，使其仅在第1位和第140位包含AS。通过在CTP和UTP存在下进行体外转录实验，并选择性地添加ATP，我们将能够确定RNA聚合酶II和核肌球蛋白I的马达活性对转录延伸的相对贡献。我们还将使用化学遗传学方法和被N6(2-甲基丁基)ADP选择性抑制的核肌球蛋白I来进一步分析核肌球蛋白I在转录中的运动功能。这些实验是对这一假设的严格和富有想象力的测试，将为转录的分子基础提供新的和重要的见解。 与公共卫生相关：转录，即将DNA转化为RNA的过程，是细胞中最基本的重要过程之一。这项建议中描述的实验研究了一种新的蛋白质在转录中的作用。它们有可能增加我们在分子水平上对转录过程的理解

DESCRIPTION (provided by applicant): Nuclear myosin I is a member of the myosin superfamily of actin activated ATPases that is found in the nucleus. There is also abundant evidence for the presence of actin in the nucleus. Nuclear myosin I and actin are necessary for transcription by RNA polymerase I and II. Specifically, actin is necessary for pre-initiation complex formation by RNA polymerase II and actin binds to RNA polymerases I, II and III. We have also found that nuclear myosin I is involved in transcription initiation and that actin and nuclear myosin I are components of elongating complexes. Based on extensive preliminary data, we propose the hypothesis that nuclear myosin I has a crucial role in transcription elongation by RNA polymerase II. Two Specific Aims are proposed to test this hypothesis. Specific Aim 1 has 2 parts. In the first part, we will analyze elongating complexes stalled on an immobilized DNA template to identify binding partners of nuclear myosin I. In the second part, we will restart paused complexes to determine the role of nuclear myosin I in elongation. Specific Aim 2 will directly investigate the possibility that actin and nuclear myosin I have a motor function in transcription elongation. Because RNA polymerase II is also a motor that hydrolyzes ATP, we re-engineered our DNA template so that it contains As only at position 1 and 140. By performing in vitro transcription assays in the presence of CTP and UTP and adding ATP selectively, we will be able to determine the relative contributions of the motor activity of RNA polymerase II and nuclear myosin I to transcription elongation. We will also use a chemical-genetic approach and a nuclear myosin I that is selectively inhibited by N6(2-methylbutyl) ADP to further analyze the motor function of nuclear myosin I in transcription. These experiments are a rigorous and imaginative test of the hypothesis that will provide new and important insights into the molecular basis of transcription. PUBLIC HEALTH RELEVANCE: Transcription, the process of converting DNA into RNA, is one of the most fundamentally important processes in cells. The experiments described in this proposal investigate the role of a new protein in transcription. They have the potential to increase our understanding of the transcription process at the molecular level