Mechanism of DEAD-box proteins

描述(申请人提供)：实际上，RNA代谢的所有方面都涉及DExH/D蛋白质，这是一大类高度保守的酶。该家族的许多蛋白质在包括肿瘤发生和传染病在内的疾病状态中发挥直接作用。DExH/D蛋白包括DEAD-box、DExH和Deah亚群，所有这三个亚群的酶都将ATP结合和/或水解与RNA解离或RNA-蛋白质复合体的结构变化联系在一起。尽管DExH/D蛋白的结构和序列是保守的，但最近清楚的是，DExH/D亚组之间存在着根本的功能差异。DEAD-box蛋白是最大的DExH/D蛋白亚群，已被发现不像以前研究的解旋酶那样通过易位来解开双链，而是通过ATP驱动的局部链分离。机制概念是为转位解旋酶而开发的，因此不适用于死盒蛋白。本文建议在分子水平上定义DEAD-box蛋白的功能。首先，将建立一个由死盒蛋白Ded1p解开RNA的动力学和热力学框架，目的是了解该蛋白如何将ATP结合和水解与RNA上的构象功结合起来。然后，这个框架将被用来分析另外两个不同的死盒蛋白，Mss 116p和eIF4A。对这三种酶进行了定量的比较分析，以深入了解DEAD-box蛋白的功能多样性。最后，作为了解DEAD-box蛋白在更复杂的生理环境中功能的第一步，我们研究了生理辅助因素如何调控Ded1P和eIF4A的机制(S)。这项拟议的研究将生物化学和生物物理学方法与单分子技术相结合。这项工作不仅对DEAD-box蛋白的分子机制提供了独特的见解，而且在概念和方法上促进了对RNA-蛋白质相互作用的定量分析和理解。DExH/D蛋白是一大类对基因表达至关重要的酶，但其功能尚不清楚。这些酶中的许多都与癌症和传染病等疾病有关。为了深入了解这些疾病的分子基础，并指导潜在治疗药物的开发，我们建议研究DExH/D蛋白的机制。

DESCRIPTION (provided by applicant): Virtually all aspects of RNA metabolism involve DExH/D proteins, a large and highly conserved class of enzymes. Numerous proteins from this family play direct roles in disease states including tumorogenesis and infectious diseases. DExH/D proteins comprise the DEAD-box, the DExH and the DEAH subgroups and enzymes from all three subgroups couple ATP binding and/or hydrolysis to RNA unwinding or structural changes in RNA-protein complexes. Despite the conservation of structure and sequence within the DExH/D proteins, it has recently become clear that fundamental functional differences exist between the DExH/D subgroups. DEAD-box proteins, the largest DExH/D protein subgroup, have been found to unwind duplexes not by translocation, like previously studied helicases, but by ATP-driven, local strand separation. Mechanistic concepts were developed for translocating helicases thus do not apply to DEAD-box proteins. Here, it is proposed to define DEAD-box protein function on the molecular level. First, a kinetic and thermodynamic framework for RNA unwinding by the DEAD-box protein Ded1p will be developed, with the aim to understand how this protein couples ATP binding and hydrolysis to conformational work on RNA. This framework will then be utilized to analyze two additional, different DEAD-box proteins, Mss116p, and eIF4A. A quantitative, comparative analysis between all three enzymes is performed, to gain insight into the functional diversity of DEAD-box proteins. Finally, as the first step towards understanding the function of DEAD-box proteins in more complex physiological environments, it is investigated how physiological co-factors modulate the mechanism(s) of Ded1p and eIF4A. The proposed study combines biochemical and biophysical ensemble methods with single molecule techniques. The work will not only provide unique insight into the molecular mechanism of DEAD- box proteins but also conceptually and methodologically advance quantitative analysis and understanding of RNA-protein interactions. DExH/D proteins are a large class of enzymes essential for gene expression, but their function is not well understood. Many of these enzymes have been implicated in disease states including cancer and infectious diseases. To provide critical insight into the molecular basis of these diseases and to guide the development of potential therapeutic agents, we propose to study the mechanism of DExH/D proteins.