Mechanism of DEAD-box proteins

描述（由申请人提供）：RNA代谢的几乎所有方面都涉及DExH/D蛋白，这是一种大型且高度保守的酶。来自该家族的许多蛋白质在包括肿瘤发生和感染性疾病的疾病状态中发挥直接作用。DExH/D蛋白包含DEAD-盒、DExH和DEAH亚组，并且来自所有三个亚组的酶将ATP结合和/或水解偶联至RNA-蛋白复合物中的RNA解旋或结构变化。尽管DExH/D蛋白质的结构和序列保守，但最近已经清楚的是，DExH/D亚组之间存在基本的功能差异。DEAD-盒蛋白是最大的DExH/D蛋白亚类，已被发现不像以前研究的解旋酶那样通过易位来解旋双链体，而是通过ATP驱动的局部链分离来解旋。因此，为易位解旋酶开发的机制概念不适用于DEAD盒蛋白。在这里，它是建议定义死亡盒蛋白质的功能在分子水平上。首先，将开发一个由DEAD盒蛋白Ded 1 p进行RNA解旋的动力学和热力学框架，目的是了解这种蛋白质如何将ATP结合和水解与RNA的构象工作相结合。然后，该框架将用于分析另外两种不同的DEAD盒蛋白，Mss 116 p和eIF 4A。所有三种酶之间的定量比较分析进行，以深入了解死亡盒蛋白质的功能多样性。最后，作为理解DEAD盒蛋白在更复杂的生理环境中的功能的第一步，研究了生理辅因子如何调节Ded 1 p和eIF 4A的机制。这项研究将生物化学和生物物理学集成方法与单分子技术相结合。这项工作不仅将为DEAD盒蛋白的分子机制提供独特的见解，而且在概念上和方法上推进定量分析和理解RNA-蛋白质相互作用。DExH/D蛋白是基因表达所必需的一大类酶，但它们的功能还不清楚。这些酶中的许多与包括癌症和传染病在内的疾病状态有关。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.