Mechansim of DExH/D proteins

描述(由申请人提供)： DExH/D蛋白几乎参与了细胞和许多病毒中RNA新陈代谢的各个方面。这个家族的许多蛋白质已被证明在疾病状态中发挥直接作用，例如在肿瘤形成以及丙型肝炎和天花病毒的复制中。DExH/D蛋白以依赖于ATP的方式解开RNA结构(RNA解旋酶)和/或重新排列RNA-蛋白质复合体(RNPase)。尽管DExH/D蛋白具有重要的生物学意义，但其作用机制尚不清楚，主要是由于反应的高度复杂性。我们建议通过使用单分子荧光结合生物化学方法来研究痘苗病毒的原型DExH/D蛋白NPH-II，从而获得对这些问题的基本见解。 首先，我们将在单分子水平上建立RNA解旋酶活性的机制框架，以解决DExH/D蛋白如何利用ATP影响RNA构象变化的根本问题。利用单分子荧光能量转移(FRET)，我们将确定ATP结合和水解是如何与RNA的构象变化、NPH-II的寡聚态以及NPH-II在双链解离过程中的移位耦合的。 其次，我们将探索NPH-II沿单链RNA的易位。有人假设，沿着单链RNA移位的能力可能导致DExH/D蛋白的多重活性，如RNA蛋白复合体的双链解离和重塑。然而，沿着单链RNA的移位从未被测试过。我们将使用单分子FRET直接检测NPH-II是否沿着单链RNA移位。 第三，我们将研究RNPase活性的物理基础。使用生化方法，我们将测试DExH/D蛋白是否通过直接物理接触取代RNA中的其他蛋白质，或通过诱导RNA中的扭转应变将其他蛋白质从RNA中移开。此外，我们还将研究NPH-II如何将ATP结合和水解耦合到RNA-蛋白质复合体的重塑中。

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.