The Structure and Reaction Dynamics of Three Catalytic RNAs

这个项目的主要焦点是生物大分子的功能如何由它们的结构来管理的问题。该研究计划在RNA酶(或核酶)的背景下关注这一问题，希望发现生物催化的特性，这些特性是如此基本，以至于它们被所有酶共享，无论是RNA还是蛋白质。这项建议的总体目标是对生物催化剂的功能有一个基本的了解，无论是蛋白质还是RNA。将使用静态和时间分辨X射线结晶学来研究两个催化RNA系统，以阐明它们的结构特性和反应机理。按照复杂性的顺序，这些包括发夹状核酶和RNaseP。锤头状核酶的结构已经被解决，时间分辨的结晶学实验已经开始。更复杂的核酶已经结晶，并将用于静态和时间分辨结晶学，以努力阐明它们的反应机理。本研究的主要目标是：(1)确定发夹状核酶的三维结构，并利用为锤头状核酶开发的技术开始测定发夹状核酶切割和连接反应中间产物的结构；(2)开始测定核糖核酶P的三维结构。这项研究的主要目标是对使RNA分子成为酶的催化原理有一个基本的了解。这些知识将有助于理解RNA酶的三维结构是如何决定其催化性能的。通过学习RNA酶的工作原理，这个项目将发现对于生物催化剂来说，什么性质是如此基本，以至于它们对于RNA酶和蛋白质酶都是不变的。

The primary focus of this project is upon the problem of how the functions of biological macromolecules are governed by their structures. The research plan focuses upon this problem in the context of RNA enzymes (or ribozymes), with the hope of discovering the properties of biological catalysis that are so fundamental that they are shared by all enzymes, whether composed of RNA or protein. The overall goal of this proposal is to develop a fundamental understanding of how biological catalysts function, be they proteins or RNA.Two catalytic RNA systems will be investigated using both static and time-resolved X-ray crystallography to elucidate their structural properties and reaction mechanisms. These, in order of increasing complexity, include the hairpin ribozyme, and RNase P. The hammerhead ribozyme structure has been solved and time-resolved crystallographic experiments have been initiated. The more complex ribozymes have been crystallized and will be used in static and time-resolved crystallography in an effort to elucidate their reaction mechanisms. The principle objectives of this research are: (1) to determine the three-dimensional structure of the hairpin ribozyme, and to initiate determination of the structures of reaction intermediates of hairpin ribozyme cleavage and ligation using the techniques developed for the hammerhead ribozyme; and (2) to initiate determination of the three-dimensional structure of RNase P.. The primary goal of this research is to develop a fundamental understanding of the principles of catalysis that enable RNA molecules to be enzymes. This knowledge will lead to the understanding of how the three-dimensional structure of an RNA enzyme determines its catalytic properties. By learning how RNA enzymes work, this project will discover what properties are so fundamental to biological catalysts that they are invariant with respect to both RNA and protein enzymes.