Mechansim of DExH/D proteins

描述（由申请人提供）： DExH/D蛋白参与细胞和许多病毒中RNA代谢的几乎所有方面。来自该家族的许多蛋白质已被证明在疾病状态如肿瘤发生和丙型肝炎病毒和天花病毒的复制中发挥直接作用。DExH/D蛋白以ATP依赖的方式解开RNA结构（RNA解旋酶）和/或重新排列RNA-蛋白质复合物（RNase）。尽管DExH/D蛋白具有生物学重要性，但其作用机制尚不清楚，主要是由于反应的高度复杂性。我们建议通过采用单分子荧光结合生物化学方法研究来自牛痘病毒的原型DExH/D蛋白NPH-II来深入了解这些问题。 首先，我们将在单分子水平上开发RNA解旋酶活性的机制框架，以解决DExH/D蛋白如何使用ATP影响RNA构象变化的基本问题。使用单分子荧光能量转移（FRET），我们将确定如何ATP结合和水解耦合到RNA的构象变化，寡聚状态的NPH-II，和双链体解旋过程中的NPH-II的易位。 第二，我们将探测NPH-Ⅱ沿着单链RNA的易位。已经假设，沿着沿着单链RNA移位的能力可以引起DExH/D蛋白的多种活性，例如双链体解旋和RNA蛋白复合物的重塑。然而，易位沿着单链RNA从未被测试过。我们将使用单分子FRET直接测试NPH-II是否沿着沿着单链RNA易位。 第三，我们将研究RNATIONAL活动的物理基础。采用生物化学方法，我们将测试DExH/D蛋白是否通过直接物理接触或通过诱导RNA中的扭转应变将其他蛋白质从RNA中置换出来。此外，我们将研究NPH-II如何将ATP结合和水解与RNA-蛋白质复合物的重塑偶联。

DESCRIPTION (provided by applicant): DExH/D proteins are involved in virtually all aspects of RNA metabolism in the cell and in many viruses. Numerous proteins from this family have been shown to play direct roles in disease states such as in tumorigenesis and in the replication of the hepatitis C and the smallpox virus. DExH/D proteins unwind RNA structures (RNA helicase) and/or re-arrange RNA-protein complexes (RNPase) in an ATP-dependent fashion. Despite the biological importance of DExH/D proteins, their mechanism of action is not understood, mainly due to the highly complex nature of the reactions. We propose to gain essential insight into these questions by employing single molecule fluorescence in conjunction with biochemical approaches to investigate the prototypical DExH/D protein NPH-II from vaccinia virus. First, we will develop a mechanistic framework for RNA helicase activity at the single molecule level to address the fundamental question the how DExH/D proteins use ATP to effect conformational changes in RNA. Using single molecule fluorescence energy transfer (FRET) we will determine how ATP binding and hydrolysis is coupled to conformational changes in the RNA, to the oligomeric state of NPH-II, and to the translocation of NPH-II during duplex unwinding. Second, we will probe translocation of NPH-II along single stranded RNA. It has been hypothesized that an ability to translocate along single stranded RNA could give rise to the multiple activities of DExH/D proteins such as duplex unwinding and remodeling of RNA protein complexes. Yet, translocation along single stranded RNA has never been tested. We will directly test whether NPH-II translocates along single stranded RNA using single molecule FRET. Third, we will investigate the physical basis of RNPase activity. Employing a biochemical approach, we will test whether DExH/D proteins displace other proteins from RNA through direct physical contact, or through induction of torsional strain in the RNA to flip other proteins off the RNA. In addition we will investigate how NPH-II couples ATP binding and hydrolysis to the remodeling of RNA-protein complexes.