Catalytic Mechanisms of RNA Ezymes

催化RNA提供了一个进入原始“RNA世界”的窗口，现代生物学可能会从中受益。 已经进化了RNA催化剂在现代蛋白质世界中的当代作用仍然存在于 调节基因表达和蛋白质合成，使RNA催化对正常生长至关重要， 发展最近的自切割和自剪接RNA的高分辨率结构提供了 锤头状、丁型肝炎病毒和发夹状自切割RNA和I组自切割RNA的活性位点， 剪接内含子。核糖体的结构使我们看到了一个肽基转移酶中心， 完全来自RNA。自第一个核酶问世以来， 现在可以与这些结构进行比较和对比，为 这些实验旨在探索RNA酶如何利用它们的官能团 催化作用作为第一个在没有金属阳离子辅因子的情况下发挥作用的核酶，发夹核酶是一种 理解非金属依赖性RNA催化的原型。在以前的奖励期间的工作定义 活性位点的基本结构和生化特征，并导致可测试的模型的作用， 催化化学中的活性中心核碱基。我们下一个奖励期的目标是确定催化剂 发夹状核酶的机制，并阐明最近发现的代谢物的机制- 依赖性核酶，其在某些关键机制特征上类似于发夹核酶。具体 目标包括1）区分发夹状核酶中的键断裂和键形成步骤的贡献 使用5'硫代磷酸酯RNA的反应，2）确定发夹核酶的微观pKa值 使用8-氮杂嘌呤荧光监测质子化中pH依赖性变化的活性位点核碱基 状态并使用异核核磁共振监测化学位移的pH依赖性变化，3）确定 单个活性中心官能团对基态和过渡态稳定性的能量贡献 将原子水平诱变与发夹状核酶复合物的物理和功能测定相结合， 形成和催化作用，以及4）建立定量分析的动力学和热力学框架， 代谢物响应性glmS核酶的反应途径。拟议的研究将产生 对RNA和RNP酶催化机制的基本见解，RNA折叠和动力学， 以及RNA与小配体的相互作用。这项工作将加深我们对RNA结构的基本认识 和功能，这是健康和疾病的基础生物学的重要组成部分。的结果予以 研究还将提供一个框架，指导技术和治疗应用的发展 涉及RNA作为靶和试剂。

Catalytic RNAs provide a window into a primordial 'RNA World' from which modern biology might have evolved. Contemporary roles for RNA catalysts in the modern protein world are still found in the regulation of gene expression and in protein synthesis, making RNA catalysis essential to normal growth and development. Recent high-resolution structures of self-cleaving and self-splicing RNAs provide pictures of the active sites for the hammerhead, hepatitis delta virus, and hairpin self-cleaving RNAs and Group-l self- splicing introns. The structure of the ribosome gave us a view of a peptidyl transferase center constructed entirely from RNA. Mechanistic studies carried out over more than 20 years since ribozymes were first discovered can now be compared and contrasted with these structures, laying the groundwork for experiments to probe fundamental questions about how RNA enzymes use their functional groups for catalysis. As the first ribozyme shown to function without metal cation cofactors, the hairpin ribozyme is a prototype for understanding metal-independent RNA catalysis. Work during previous award periods defined the essential structural and biochemical features of the active site and led to testable models for the roles of active site nucleobases in catalytic chemistry. Our goal for the coming award period is to define the catalytic mechanism of the hairpin ribozyme and elucidate the mechanisms of a recently discovered metabolite- dependent ribozyme, which resembles the hairpin ribozyme in certain key mechanistic features. Specific goals include 1) distinguishing contributions to bond breaking and bond making steps in the hairpin ribozyme reaction using 5' phosphorothiolate RNAs, 2) determining the microscopic pKa values of hairpin ribozyme active site nucleobases using 8-azapurine fluorescence to monitor pH-dependent changes in protonation states and using heteronuclear NMR to monitor pH-dependent changes in chemical shifts, 3) determine the energetic contributions of individual active site functional groups to ground state and transition state stability by combining atomic level mutagenesis with physical and functional assays of hairpin ribozyme complex formation and catalysis, and 4) establish a kinetic and thermodynamic framework for quantitative analysis of the reaction pathway of the metabolite-responsive glmS ribozyme. The proposed studies will generate fundamental insights into mechanisms of catalysis by RNA and RNP enzymes, RNA folding and dynamics, and RNA interactions with small ligands. This work will deepen our basic understanding of RNA structure and function, which is a critical part of the fundamental biology of health and disease. The results of these studies also will provide a framework to guide the development of technical and therapeutic applications involving RNAs as targets and reagents.