Catalytic Mechanisms of RNA Ezymes

催化RNA为我们打开了一扇通往原始‘RNA世界’的窗口，现代生物学可能会从这个世界出发 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催化对正常生长和 发展。最近的自切割和自剪接RNA的高分辨率结构提供了 锤头状病毒、丁型肝炎病毒和发夹状自裂解RNA的活性部位和群-L自体 拼接内含子。核糖体的结构使我们看到了构建的肽基转移酶中心。 完全来自核糖核酸。自核酶首次被发现以来，已进行了20多年的机制研究 现在可以与这些结构进行比较和对比，为 探索关于RNA酶如何利用它们的官能团来 催化作用。作为第一个在没有金属阳离子辅助因子的情况下发挥作用的核酶，发夹状核酶是一种 理解金属非依赖的RNA催化的原型。在以前定义的奖励期间内的工作 活性部位的基本结构和生化特征，并导致了可测试的模型的作用 催化化学中的活性中心碱基。我们在接下来的获奖期的目标是定义催化剂 发夹状核酶的作用机制以及新近发现的一种代谢物-- 依赖核酶，在某些关键的机制特征上类似于发夹核酶。特定的 目标包括1)区分发夹核酶中断键和成键步骤的贡献 5‘硫代磷酸核糖核酸的反应，2)发夹状核酶的显微pKa值的测定 用8-氮杂嘌呤荧光监测质子化过程中与pH相关的碱基 并使用异核核磁共振监测化学位移随pH的变化，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 通过原子水平突变与发夹状核酶复合体的物理和功能分析相结合 形成和催化，以及4)建立了定量分析的动力学和热力学框架 代谢物反应型GLMS核酶的反应途径。拟议的研究将产生 对RNA和RNP酶的催化机制、RNA折叠和动力学的基本见解， 以及与小配体的RNA相互作用。这项工作将加深我们对RNA结构的基本理解 和功能，这是健康和疾病的基础生物学的关键部分。这些研究的结果 研究还将提供一个框架，指导技术和治疗应用的开发 将RNA作为靶标和试剂。

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.