Mechanism of RNA helicases

RNA解旋酶是真核生物RNA代谢中最大的一类酶，具有高度保守性， 在几乎所有细胞RNA交易中起重要作用。许多RNA的突变和失调 解旋酶与包括癌症、病毒感染和神经变性在内的疾病状态有关 紊乱尽管这些酶具有关键的生物学作用，但目前尚不清楚绝大多数酶是如何被激活的。 RNA解旋酶在分子水平上发挥生理反应。 两个主要问题阻碍了对生物化学反应的生物化学机制的明确定义， RNA解旋酶。首先，尚不清楚其他蛋白质和辅助因子如何影响生物化学， RNA解旋酶的功能其次，几乎所有的RNA解旋酶都不知道在哪里结合它们的生物活性。 印刷受体.蛋白质，其中大多数不是固有的序列或结构特异性的，因此不能被识别。 物理连接到它们的基底上。 在这个提议中，我们直接解决了RNA解旋酶领域的这两个长期存在的问题。我们 重点研究酿酒酵母DEAD-box RNA解旋酶Ded 1 p及其在翻译中的功能 入会仪式在目的1中，我们将确定Ded 1 p的生化特性以及与细胞因子的相互作用如何影响细胞的功能。 辅因子eIF 4G 1影响Ded 1 p在翻译起始中的功能，使用分子生物学结合 以及定量生物化学和生物物理方法。在目标2中，我们将定义生理RNA结合 位点的Ded 1 p，使用基于交联和下一代测序的系统方法。我们 计划进一步分析Ded 1 p与其mRNA同源位点结合的功能意义， 分子生物学和生物化学方法。我们希望我们的研究能提供基本的新见解 RNA解旋酶的分子功能。

RNA helicases, the largest class of enzymes in eukaryotic RNA metabolism, are highly conserved and essential players in almost all cellular RNA transactions. Mutations and deregulation of numerous RNA helicases have been linked to disease states including cancer, viral infections, and neurodegenerative disorders. Despite the pivotal biological roles of these enzymes, it is not known how the vast majority of RNA helicases exerts physiological reactions at the molecular level. Two major problems prevent clear definitions of biochemical mechanisms for physiological reactions of RNA helicases. First, it is not well understood how other proteins and co-factors affect biochemical functions of RNA helicases. Second, it is not known where almost all RNA helicases bind their biological substrates. The proteins, most of which are not inherently sequence or structure specific, thus cannot be physically linked to their substrates. In this proposal, we directly address both of these longstanding problems in the RNA helicase field. We focus on the DEAD-box RNA helicase Ded1p from Saccharomyces cerevisiae and its function in translation initiation. In Aim 1 we will determine how biochemical characteristics of Ded1p and the interaction with the co-factor eIF4G1 affect Ded1p's function in translation initiation, using a combination of molecular biological and quantitative biochemical and biophysical approaches. In Aim 2 we will define physiological RNA binding sites of Ded1p, using a systematic approach based on crosslinking and next generation sequencing. We plan to further analyze the functional significance of Ded1p binding to its cognate sites on mRNAs using molecular biology and biochemical approaches. We expect our studies to provide fundamental new insight into molecular functions of RNA helicases.