ENZYMOLOGY OF RNA PROCESSING ENZYMES

RNA 加工酶的酶学

• 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
• 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

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的必需5'成熟。 而两个 蛋白质和RNA亚基对于体内活性至关重要 RNase P RNA部分在体外表现出催化活性。 机械和 该核酶的结构研究很重要，因为它是 只有已知的含RNA的核酸酶，可催化多个失误，而IS 催化过程中没有变化。 该建议概述了一项协作研究 Fierke和Christianson团体之间的努力探究酶学 和枯草芽孢杆菌RNase P.催化效率的结构生物学将是 通过X射线组合与大分子结构有关 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 枯草芽孢杆菌的蛋白质成分的三维结构 RNase P; （2）研究RNase P和3'的催化机制， 使用稳态和瞬态状态的DNA聚合酶I的5'外切酶 动力学探测催化对金属和磷酸盐酸盐的依赖性 替代，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 RNase P的组成部分通过动力学，足迹， 交联和诱变； （4）确定 通过修饰解干实验的必需镁离子和 P RNA中的磷酸盐剂取代； （5）选择与 RNase P蛋白来自RNase P RNA片段的库和 完全随机的RNA，用于研究RNA蛋白的结构 X射线晶体学的复合物； （6）探索A的结晶 催化活性RNase P全酶，尤其是使用圆形RNA 组件。这些提出的RNase的机械和结构功能研究 p将进一步了解核酶中使用的催化模式 与蛋白质催化剂的比较，RNA结构与 功能，以及RNA-RNA和RNA-蛋白的特定相互作用 结合。 对体外原理的基本理解 实验是理解体内RNA催化的先决条件，因为 设计基于机制的药物以抑制RNase P和核酶， 对于工程内核核酸的特定于特定的RNA物种， 包括核酶的治疗用途。 此外，由于RNase P是 该酶是一个必需的细菌酶，可能代表可行靶标 用于新型抗生素。