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(RNaseP)是一种核糖核蛋白。 催化前体tRNA的基本5‘成熟。虽然两个人都 蛋白质和RNA亚基对于体内的活动是必不可少的，细菌 核糖核酸酶P-RNA部分在体外具有催化活性。机械和 这种核酶的结构研究很重要，因为它是 唯一已知的含有RNA的核酸酶，它催化多次转换，并且 在催化过程中保持不变。这份提案概述了一项合作研究 费尔克和克里斯汀森小组为探索酶学所做的努力 而枯草杆菌核糖核酸酶P.的结构生物学催化效率 通过X射线的组合与大分子结构相关 结晶学、动力学和平衡分析以及结构 微扰。具体地说，我们的目标是：(1)确定和完善 枯草芽孢杆菌蛋白质组分的三维结构 RNaseP；(2)研究RNaseP和3‘的催化机理， 稳态法和瞬态法测定DNA聚合酶I的5‘外切酶 探讨催化剂对金属和硫代磷的依赖性的动力学 取代度、pH、溶剂的同位素组成和结构 酶的变异体；(3)研究蛋白质的功能作用 RNaseP的组成成分通过动力学、足迹、 交联剂和诱变剂；(4)确定 必需镁离子的改性-干扰实验和 P-RNA中的硫代磷酸取代；(5)选择与其结合的RNA 从RNase P RNA和RNase P RNA片段文库中获得RNase P蛋白 全随机RNA，用于研究RNA-蛋白质的结构 X-射线结晶学分析；以及(6)探索一种新化合物的结晶 催化活性核糖核酸酶P全酶，特别是使用环状RNA 提出了核糖核酸酶的作用机制和结构-功能研究 P将进一步加深我们对核酶在体内的催化模式的理解 与蛋白质催化剂相比，RNA结构与蛋白质之间的关系 功能，以及RNA-RNA和RNA-蛋白质所涉及的特定相互作用 有约束力的。从体外对这些原理的基本理解 实验是理解体内RNA催化作用的前提，因为 设计基于机制的药物来总体上抑制RNaseP和核酶， 并且对于针对特定RNA物种的工程内切核酸酶， 包括核酶的治疗用途。此外，由于RNaseP是 作为一种重要的细菌酶，这种酶可能是一个可行的靶标。 寻找新的抗生素。