Mechanism of RNA helicases

RNA解旋酶是真核细胞RNA代谢中最大的一类酶，具有高度保守性和 在几乎所有的细胞RNA交易中扮演着重要的角色。众多核糖核酸的突变和解除调控 解旋酶与癌症、病毒感染和神经退行性变等疾病状态有关。 精神错乱。尽管这些酶具有关键的生物学作用，但尚不清楚绝大多数酶是如何 RNA解旋酶在分子水平上进行生理反应。 两个主要问题阻碍了对生理学反应的生化机制的明确定义 RNA解旋酶。首先，人们还不清楚其他蛋白质和辅助因素是如何影响生化的。 RNA解旋酶的功能。其次，目前还不知道几乎所有的RNA解旋酶在哪里结合了它们的生物 底物。这些蛋白质大多数本身并不是序列或结构特定的，因此不可能是 在物理上与它们的底物相连。 在这项提案中，我们直接解决了RNA解旋酶领域中的这两个长期存在的问题。我们 酿酒酵母Dead-box RNA解旋酶Ded1p及其在翻译中的作用 入会仪式。在目标1中，我们将确定Ded1p的生化特性以及与Ded1p的相互作用 辅因子eIF4G1影响Ded1p在翻译起始中的功能 以及定量的生化和生物物理方法。在目标2中，我们将定义生理性RNA结合 Ded1p的位点，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Ded1p与其在mRNAs上的同源位点结合的功能意义 分子生物学和生化方法。我们希望我们的研究能提供根本性的新见解 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.