Coupling of structure and dynamics in RNA catalysis

描述(由申请人提供)：本提案的主要目标是从分子水平了解核酶(核酶)如何催化化学反应。我们正在研究一种自我裂解的RNA，它最初是在人类丁型肝炎病毒中发现的，但现在已知在自然界中广泛分布。这种核酶利用具有显着移位的PKA和二价金属离子的核苷酸碱基来催化RNA切割反应。我们将结合X射线结晶学、分子动力学和溶液生物化学实验来了解RNA的三维结构如何与其金属辅因子相互作用来实现催化，以及这种动态RNA的分子运动如何有助于其反应活性。我们的第一个具体目标描述了我们将使用X射线结晶学来解决三维结构的策略，并验证晶体中观察到的构象和任何无序都模仿溶液中活性核酶的构象。在我们的第二个具体目标中，我们将使用分子动力学来描述核酶活性部位内发生的运动，并了解无序在核酶催化中的作用。最后一个具体目标描述了旨在剖析活性中心组分对催化的贡献的生化和光谱实验。我们将分析催化金属离子的潜在配体，探索催化金属离子促进催化的机理，并利用溶液生物化学探索核苷酸在剪刀状磷酸根上游的定位和运动以及它们对反应途径的贡献。这项研究的结果将提供一种核酶的深入结构和机制分析。然而，在后基因组时代，我们看到了非编码RNA序列对基因表达调控的意外贡献。核酶和核糖开关在各种环境中被发现，包括在真核转录本中。因此，在我们的知识库中有一些深入的知识，如丁型肝炎病毒核酶，以充分了解常见核酶和独特的孤儿核酶的催化潜力是必要的。 公共卫生相关性：在后基因组时代，我们看到非编码RNA序列，包括核酶，对原核和真核基因表达的调控做出了意想不到的贡献。为了充分了解这些非编码的功能RNA是如何工作的，我们正在对核酶进行深入的结构和机制分析。我们预计，这项研究的结果将为许多核酶的催化策略提供线索，其中一些核酶将成为治疗靶点。

DESCRIPTION (provided by applicant): The broad goals of this proposal are to provide a molecular-level understanding of how RNA enzymes (ribozymes) catalyze chemical reactions. We are studying a self-cleaving RNA that was originally identified in the human hepatitis delta virus, but is now known to be widely distributed in nature. This ribozyme harnesses a nucleobase with a dramatically shifted pKA and a divalent metal ion to catalyze an RNA cleavage reaction. We will integrate X-ray crystallography, molecular dynamics, and solution biochemistry experiments to learn how the three dimensional structure of the RNA interacts with its metal cofactors to achieve catalysis and how the molecular motions of this dynamic RNA contribute to its reactivity. Our first specific aim describes the strategies we will use to solve the three dimensional structure using X-ray crystallography, and to verify that the conformation and any disorder observed in the crystal mimics the conformation of the active ribozyme in solution. In our second specific aim, we will use molecular dynamics to characterize the motions that occur within the ribozyme active site and to understand the role of disorder in ribozyme catalysis. The last specific aim describes biochemical and spectroscopic experiments designed to dissect the contributions of active site components to catalysis. We will analyze potential ligands to the catalytic metal ion, probe the mechanism by which the catalytic metal ion contributes to catalysis, and explore using solution biochemistry the positioning and motions of nucleotides upstream of the scissile phosphate and how they contribute to the reaction pathway. The results of this study will provide an in-depth structural and mechanistic analysis of one ribozyme. However, in the post genomic age, we are seeing an unexpected contribution of non-coding RNA sequences to regulation of gene expression. Ribozymes and riboswitches are being discovered in a variety of contexts, including within eukaryotic transcriptomes. It is therefore essential to have in our knowledge base some in-depth knowledge of a few paradigm systems such as the hepatitis delta virus ribozyme in order to fully understand the catalytic potential of common ribozymes and unique orphan ribozymes. PUBLIC HEALTH RELEVANCE: In the post genomic age, we are seeing an unexpected contribution of non-coding RNA sequences, including ribozymes, to regulation of prokaryotic and eukaryotic gene expression. To fully understand how these non-coding, functional RNAs work, we are undertaking an in depth structural and mechanistic analysis of a ribozyme. We anticipate that the results of this study will provide clues as to the catalytic strategies of many ribozymes, some of which will be therapeutic targets.