Structure and Catalysis of an RNA Enzyme

描述(申请人提供)：这里提出的这项研究的总体目标是了解一种名为锤头状核酶的小RNA酶如何工作的根本问题。利用静态X射线结晶学、动态结晶学中间捕获实验(一种时间分辨结晶学)，再加上固态核磁共振协作和各种生化方法，将以各种方式测试RNA分子本身将以各种方式测试，而不是简单地作为相对被动的金属离子结合支架，而是积极参与催化化学。通过这样做，将获得对催化RNA结构和功能(即催化)之间的关系的理解。本研究计划的具体目的是：(1)回答锤头状核酶如何能够在RNA内切酶和RNA连接酶之间切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催化所需构象变化的作用力；(3)解决新发现的锤头状核酶结构，该结构在螺旋茎I和II之间具有稳定的三级结构接触，其催化速率比先前研究的锤头状RNA序列提高1000倍；(4)通过设计新的催化RNA来检验我们对核酶催化的理解。这四个特定目的中的每一个都是为了从不同的角度独立地探索锤头状核酶的切割机制。锤头状核酶作为针对RNA病毒(如HIV)和病理性mRNAs(如癌基因转录本)的潜在治疗剂的潜在用途已得到很好的证明。虽然我们在这里提出的研究的主要动机是回答基本的科学性质的问题，但希望这些研究的结果将为科学界和医学界提供实用信息，使其他人能够开发更有效和更有效的核酶药物。

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.