Structure and Catalysis of an RNA Enzyme

描述(由申请人提供)：研究的全球目标 在这里提出的是理解一个基本的问题，即一个小的RNA如何 一种名为锤头状核酶的酶起作用。使用静态X射线 结晶学，动态结晶学中间捕获实验(a 时间分辨结晶学的形式)，以及固态核磁共振 合作和各种生化方法，假设 RNA分子本身，而不是简单地充当相对被动的 结合金属离子的支架，积极参与化学 催化作用，将以各种方式进行测试。在这样做的过程中，一种理解 催化RNA结构和功能之间的关系(即， 催化)将会被获得。本文件中描述的计划的具体目标 研究建议是：(1)阐明推动和稳定经济增长的力量 已知的锤头蛋白催化所需的构象变化 核酶结构；(2)推导锤头状核酶的结构 过渡态；(3)确证其催化活性 结晶锤头状核酶更好地理解其作用机制 以前在我们的时间分辨中观察到的结构变化的含义 结晶学实验；以及(4)测试我们对核酶的理解 通过设计新的催化RNA进行催化。这四个具体目标中的每个都是 被设计成独立地探测锤头的分裂机制 来自不同观点的核酶。锤头的潜在用途 核酶作为靶向RNA病毒(如艾滋病毒)和 病理性的mRNAs(如癌基因转录本)是有充分证据的。虽然 我们在这里提出的研究的主要动机是回答一个 基本的科学性，希望这些研究的结果 将向科学界和医学界提供实用信息 使更有效和更有效的核酶药物得以开发 被其他人。

DESCRIPTION (provided by applicant): The global objective of the research proposed here is to understand the fundamental question of how a small RNA enzyme, called the hammerhead ribozyme, works. Using static X-ray crystallography, dynamic crystallographic intermediate trapping experiments (a form of time-resolved crystallography), together with a solid-state NMR collaboration and a variety of biochemical approaches, the hypothesis that that the RNA molecule itself, rather than simply acting as a relatively passive scaffold for binding metal ions, actively participates in the chemistry of catalysis, will be tested in a variety of ways. In doing so, an understanding of the relationship between catalytic RNA structure and function (i.e., catalysis) will be obtained. The specific aims of the program described in this research proposal are: (1) to elucidate the forces that drive and stabilize the conformational change known to be required for catalysis in the hammerhead ribozyme structure; (2) to deduce the structure of the hammerhead-ribozyme transition-state; (3) to characterize definitively the catalytic activity of the crystallized hammerhead ribozyme to better understand the mechanistic implications of the structural changes previously observed in our time-resolved crystallographic experiments; and (4) to test our understanding of ribozyme catalysis by designing new catalytic RNAs. Each of these four specific aims is designed to probe, independently, the cleavage mechanism of the hammerhead ribozyme from a variety of viewpoints. The potential use of hammerhead ribozymes as therapeutic agents that target RNA viruses (such as HIV) and pathological mRNAs (such as oncogene transcripts) is well-documented. Although our primary motive for the research proposed here is to answer questions of a fundamental scientific nature, it is hoped that the results of these studies will provide practical information to the scientific and medical communities to enable more potent and effective ribozyme-based pharmaceuticals to be developed by others.