ENZYMOLOGY OF RNA PROCESSING ENZYMES

描述：核糖核酸酶P（RNase P）是一种核糖核蛋白， 催化前体tRNA的必要5'成熟。 虽然双方 蛋白质和RNA亚基是体内活性所必需的， 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是重要的，因为它是 只有已知的含RNA的核酸酶催化多个周转， 在催化过程中保持不变。 该提案概述了一项合作研究 Fierke和Christianson小组为探索酶学所做的努力 和B的结构生物学枯草杆菌RNase P。催化效率将是 与大分子结构相关的X射线 晶体学、动力学和平衡分析，以及结构 扰动 具体而言，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 （2）研究了RNase P和3 '， 使用稳态和瞬态的DNA聚合酶I的5'外切核酸酶 动力学探测催化对金属和硫代磷酸酯的依赖性 取代度、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 通过动力学，足迹法， 交联和诱变;（4）确定的位置和作用， 通过修饰干扰实验测定必需镁离子， （5）选择结合于PRNA中的硫代磷酸酯取代的RNA; 来自RNase P RNA片段文库的RNase P蛋白， 完全随机的RNA，用于研究RNA蛋白质的结构 复合物的X射线晶体学;和（6）探索结晶的一个 催化活性的RNase P全酶，特别是使用环状RNA 这些建议的机制和结构功能的研究核糖核酸酶 P将进一步我们的理解所使用的催化模式的核酶， 与蛋白质催化剂相比，RNA结构与 功能，以及RNA-RNA和RNA-蛋白质之间的特异性相互作用 约束力 从体外对这些原理的基本理解 实验是理解体内RNA催化的先决条件， 设计基于机制的药物来抑制RNA酶P和核酶， 以及用于工程化特异于特定RNA种类的核糖核酸内切酶， 包括核酶的治疗用途。 此外，由于RNase P 一种必需的细菌酶，这种酶可能是一种可行的靶点， 新型抗生素。

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.