ENZYMOLOGY OF RNA PROCESSING ENZYMES

RNA 加工酶的酶学

基本信息

批准号：
6044649
负责人：
CAROL A FIERKE
金额：
$ 5.78万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-01-01 至 1999-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6044649
关键词：
Bacillus subtilis DNA footprinting RNA binding protein RNA directed DNA polymerase X ray crystallography crosslink endoribonucleases enzyme activity enzyme mechanism enzyme structure exonuclease magnesium ion nuclear magnetic resonance spectroscopy ribonucleoproteins ribozymes structural biology

项目摘要

DESCRIPTION: Ribonuclease P (RNase P ) is a ribonucleoprotein that catalyzes the essential 5' maturation of precursor tRNA. While both the protein and RNA subunits are essential for in vivo activity, the bacterial RNase P RNA moiety exhibits catalytic activity in vitro. Mechanistic and structural investigations of this ribozyme are important because it is the only known RNA-containing nuclease that catalyzes multiple turnovers and is unchanged during catalysis. This proposal outlines a collaborative research effort between the Fierke and Christianson groups to probe the enzymology and structural biology of B. subtilis RNase P. Catalytic efficiency will be related to macromolecular structure through a combination of X-ray crystallography, kinetic and equilibrium analysis, and structural perturbation. Specifically, we aim to: (1) determine and refine the three-dimensional structure of the protein component of Bacillus subtilis RNase P; (2) investigate the catalytic mechanism of both RNase P and the 3', 5' exonuclease of DNA polymerase I using steady state and transient state kinetics to probe the dependence of catalysis on metal and phosphorothioate substitution, pH, isotopic composition of the solvent, and structural variants of the enzyme; (3) investigate the functional role of the protein component of RNase P by a combination of kinetics, footprinting, crosslinking and mutagenesis; (4) identify the position and role of essential magnesium ions by modification-interference experiments and phosphorothioate substitutions in the P RNA; (5) select RNAs that bind to the RNase P protein from libraries of fragments of RNase P RNA and completely random RNA, for use in investigating the structure of RNA-protein complexes by X-ray crystallography; and (6) explore the crystallization of a catalytically-active RNase P holoenzyme, especially using a circular RNA component.These proposed mechanistic and structure-function studies of RNase P will further our understanding of the catalytic modes used by ribozymes in comparison to protein catalysts, the relationship between RNA structure and function, and the specific interactions involved in RNA-RNA and RNA-protein binding. A fundamental understanding of these principles from in vitro experiments is a prerequisite for comprehending RNA catalysis in vivo, for designing mechanism-based drugs to inhibit RNase P and ribozymes in general, and for engineering endoribonucleases specific for particular RNA species, including the therapeutic use of ribozymes. Furthermore, since RNase P is an essential bacterial enzyme, this enzyme may represent a feasible target for novel antibiotics.

描述：核糖核酸酶 P (RNase P) 是一种核糖核蛋白，催化前体 tRNA 必需的 5' 成熟。虽然两者都蛋白质和RNA亚基对于体内活动至关重要，细菌 RNase P RNA 部分在体外表现出催化活性。机械和这种核酶的结构研究很重要，因为它是唯一已知的含 RNA 的核酸酶，可催化多次转换，并且是催化过程中不变。该提案概述了一项合作研究 Fierke 和 Christianson 小组合作探索酶学枯草芽孢杆菌 RNase P 的结构生物学和结构生物学。催化效率为通过结合X射线与大分子结构相关晶体学、动力学和平衡分析以及结构扰动。具体来说，我们的目标是：（1）确定并完善枯草芽孢杆菌蛋白质成分的三维结构核糖核酸酶P； (2) 研究RNase P和3'的催化机制，使用稳态和瞬态的 DNA 聚合酶 I 5' 核酸外切酶动力学探讨催化对金属和硫代磷酸酯的依赖性取代、pH、溶剂的同位素组成和结构酶的变体； (3)研究蛋白质的功能作用通过结合动力学、足迹、交联和诱变； (4)明确岗位和作用通过修饰干扰实验获得必需的镁离子 P RNA 中的硫代磷酸酯取代； (5) 选择结合的RNA 来自 RNase P RNA 片段文库的 RNase P 蛋白和完全随机的RNA，用于研究RNA-蛋白质的结构通过 X 射线晶体学分析复合物； (6) 探索结晶催化活性 RNase P 全酶，特别是使用环状 RNA 这些提出的 RNase 机制和结构功能研究 P 将进一步加深我们对核酶催化模式的理解与蛋白质催化剂相比，RNA结构与功能，以及 RNA-RNA 和 RNA-蛋白质中涉及的特定相互作用绑定。从体外对这些原理有基本的了解实验是理解 RNA 体内催化作用的先决条件设计基于机制的药物来抑制 RNase P 和核酶，以及用于工程化针对特定 RNA 种类的内切核糖核酸酶，包括核酶的治疗用途。此外，由于 RNase P 是一种重要的细菌酶，这种酶可能代表一个可行的目标用于新型抗生素。