Functional Characterization of Nuclear Myosin I in Transcription

描述（由申请人提供）：核肌凝蛋白I是肌动蛋白激活atp酶的肌凝蛋白超家族的成员，存在于细胞核中。也有大量证据表明肌动蛋白存在于细胞核中。核肌凝蛋白I和肌动蛋白是RNA聚合酶I和II转录所必需的。具体来说，肌动蛋白是RNA聚合酶II形成起始前复合物所必需的，肌动蛋白与RNA聚合酶I、II和III结合。我们还发现核肌凝蛋白I参与转录起始，肌动蛋白和核肌凝蛋白I是拉长复合体的组成部分。基于广泛的初步数据，我们提出核肌凝蛋白I在RNA聚合酶II的转录延伸中起关键作用的假设。提出了两个具体目标来检验这一假设。具体目标1有两个部分。在第一部分中，我们将分析停滞在固定DNA模板上的延长复合物，以识别核肌凝蛋白I的结合伙伴。在第二部分中，我们将重新启动暂停的复合物，以确定核肌凝蛋白I在延长中的作用。特异性目的2将直接研究肌动蛋白和核肌球蛋白1在转录伸长中具有运动功能的可能性。由于RNA聚合酶II也是一种水解ATP的马达，我们重新设计了我们的DNA模板，使其仅在位置1和140处含有As。通过在CTP和UTP存在的情况下进行体外转录分析，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的运动活性对转录伸长的相对贡献。我们还将使用化学遗传学方法和选择性被N6（2-甲基丁基）ADP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。

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