Structure and Catalysis of an RNA Enzyme

描述（由申请人提供）：这里提出的研究的全球目标是了解一种称为锤头状核酶的小RNA酶如何工作的基本问题。使用静态X射线晶体学，动态晶体学中间体捕获实验（时间分辨晶体学的一种形式），以及固态NMR合作和各种生物化学方法，RNA分子本身，而不是简单地作为结合金属离子的相对被动的支架，积极参与催化化学的假设，将以各种方式进行测试。在这样做的过程中，理解催化RNA结构和功能之间的关系（即，催化剂）将获得。本研究计划的具体目标是：（1）回答锤头状核酶如何能够在RNA内切酶和RNA连接酶之间转换其催化活性的问题;（2）阐明驱动和稳定锤头状核酶结构中已知催化所需的构象变化的力;（3）解析新发现的锤头状核酶构建体的结构，该构建体在螺旋茎I和II之间具有稳定的三级结构接触，与先前研究的锤头状RNA序列相比，其催化速率增加了1000倍，以及（4）通过设计新的催化RNA来测试我们对核酶催化的理解。这四个特定目标中的每一个都被设计成从各种观点独立地探测锤头状核酶的切割机制。锤头状核酶作为靶向RNA病毒（如HIV）和病理性mRNA（如致癌基因转录物）的治疗剂的潜在用途已有充分记载。虽然我们在此提出的研究的主要动机是回答基础科学性质的问题，但希望这些研究的结果将为科学和医学界提供实用信息，使其他人能够开发出更有效的基于核酶的药物。

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 answer the question of how the hammerhead ribozyme is able to switch its catalytic activity between that of an RNA endonuclease and that of an RNA ligase; (2) to elucidate the forces that drive and stabilize the conformational change known to be required for catalysis in the hammerhead ribozyme structure; (3) to solve the structure of a newly discovered hammerhead ribozyme construct that possesses stabilizing tertiary structural contacts between helical Stems I and II which increase its catalytic rate 1000-fold compared to previously studied hammerhead RNA sequences, 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.