Mechanism of RNA Helicase Activity by DExH/D Proteins

描述（由申请人提供）NS 3解旋酶是丙型肝炎病毒（HCV）复制机制的重要组成部分，丙型肝炎病毒是一种正链RNA病毒，对公众健康构成重大威胁。NS 3是一种多功能分子马达，在DNA和RNA双链体解旋期间水解核苷酸三磷酸。它是解旋酶超家族2（SF 2）的成员，属于DExH/D酶亚组，参与RNA代谢的所有方面，包括前mRNA剪接、RNA干扰、翻译、RNA降解和许多形式的病毒复制。尽管DExH/D酶的普遍存在，它们对高等生物的生存能力的根本重要性，以及它们在许多人类病原体中的作用，但很少有关于RNA解旋和核糖核蛋白重塑的分子机制的研究。关于它们的核酸特异性、它们的移位模式、链置换机制或解旋期间ATP水解和功消耗之间的耦合知之甚少。为了解决这些问题，我们建议使用NS 3作为探索DExH/D蛋白行为的模型系统。这是一个很好的原型，原因有很多：（a）。NS 3已被结构表征，并且它是DExH/D成员的遗传学典型，（B）。它是一个大型核糖核蛋白机器的一部分，该机器包含调节其行为的辅因子（如大多数DExH/D蛋白），（c）。它有一个强大的解旋反应，已经定性研究，（d）。关于NS 3的信息可能对HCV管理策略的发展至关重要。我们提出了一个全面的程序NS 3解旋酶活性的生物物理特性。我们将研究分离的NS 3酶的行为及其与调节辅因子NS 4A和NS 5 B复合的特性。NS 3的DNA解旋也将被探索，因为该反应在机制上是不同的，它可能为病毒的代谢策略提供重要的见解。实验将通过应用新的预稳态动力学方法来进行，该方法监测组合底物库的解旋。这种方法，加上偶联的ATP酶测定、对化学修饰的双链体底物的研究、时间分辨足迹和单分子方法，将使我们能够表征NS 3在各种大分子环境中的微观行为。这些结果将从根本上扩展我们对解旋酶机制的认识，并促进新的HCV抑制剂的开发。

DESCRIPTION (provided by applicant) The NS3 helicase is an essential constituent of the replication machinery from Hepatitis C Virus (HCV), which is a positive-strand RNA virus that represents a major threat to public health. NS3 is a multifunctional molecular motor that hydrolyzes nucleotide triphosphates during the unwinding of both DNA and RNA duplexes. It is a member of helicase superfamily 2 (SF2), and it belongs to the DExH/D subgroup of enzymes that are involved in all aspects of RNA metabolism, including pre-mRNA splicing, RNA interference, translation, RNA degradation, and in many forms of viral replication. Despite the ubiquity of the DExH/D enzymes, their fundamental importance for viability of higher organisms, and their role in numerous human pathogens, there have been few studies on the molecular mechanisms for RNA unwinding and ribonucleoprotein remodeling by this family of proteins. Little is known about their nucleic acid specificity, their mode of translocation, the mechanism of strand displacement, or the coupling between ATP hydrolysis and work expended during unwinding. To address these issues, we propose to use NS3 as a model system for exploring the behavior of DExH/D proteins. It is an excellent prototype for numerous reasons: (a). NS3 has been structurally characterized and it is a phylogenetically typical of DExH/D member, (b). It is part of a large ribonucleoprotein machine that contains cofactors that modulate its behavior (like most DExH/D proteins), (c). It has a robust unwinding reaction that has been investigated qualitatively, (d). Information about NS3 is likely to be critical for the development HCV management strategies. We propose a comprehensive program for the biophysical characterization of NS3 helicase activity. We will examine behavior of the isolated NS3 enzyme and its properties in complex with modulatory cofactors NS4A and NS5B. DNA unwinding by NS3 will also be explored, as the reaction is mechanistically distinct and it may provide important insights into metabolic strategies of the virus. Experiments will be carried out by applying new pre-steady state kinetic methods that monitor the unwinding of combinatorial substrate libraries. This approach, together with coupled ATPase assays, studies on chemically modified duplex substrates, time-resolved footprinting and single molecule methods will allow us to characterize the microscopic behavior of NS3 in a variety of macromolecular contexts. The results will fundamentally extend our knowledge of helicase mechanism and facilitate the development of new HCV inhibitors.