CATALYTIC MECHANISM OF THE HAIRPIN RIBOZYME

描述：这项工作的长期目标是阐明 小RNA酶的催化作用。 分子间发夹的详细分析 锤头状核酶反应显示，这两种催化RNA使用 不同的动力学机制，最显着的区别是 发夹状核酶催化RNA连接的倾向。 pH和 切割和连接动力学的二价金属阳离子独立性还 指出了发夹状核酶独特的催化机制。 的 研究人员建议开发和测试发夹的详细模型 核酶催化化学 去问宇宙中最高的能量屏障 发夹断裂是破坏磷和5'氧之间的键， 研究者将测定发夹的裂解速率常数 底物，其中反应性磷酸二酯已被取代， 5 '-桥接硫代磷酸酯。 因为硫磺是一种更好的 基团比氧，预期桥连5'氧的硫取代 来加速磷硫键的断裂 如果这一步 速率决定，硫酯的裂解将比硫酯的裂解更快。 天然基质。 因为金属氢氧化物作为一般的 发夹催化中的碱基已被排除，研究人员将检查 核酶中用于催化质子的能力的官能团 转移 异源NMR实验将用于鉴定功能性化合物。 可能表现出pK值偏移的基团。 增强对以下方面的敏感性 亲电试剂的共价修饰可能揭示了一种潜在的 发夹状核酶RNA内的一般碱基催化剂。 核苷酸碱基 可能作为一般酸或碱催化剂的催化剂将被碱取代 pKa值改变的类似物。 催化剂的pH依赖性变化 引入修饰的核苷酸所产生的速率常数 具有改变的pKa值的碱将提供强有力的证据， 以催化化学为基础 调查人员将尝试识别 基态相互作用，反映了一个不寻常的电离状态， 核苷酸碱基 最后，研究人员将探讨为什么发夹 核酶是一种比核酸酶更好的连接酶， 热力学参数的内部平衡之间的解理和 连接，并通过评估三级相互作用在稳定 核酶-枯草芽孢杆菌复合物。

DESCRIPTION: The long term goal of the work is to elucidate mechanisms of catalysis by small RNA enzymes. Detailed analyses of intermolecular hairpin an hammerhead ribozyme reactions revealed that the two catalytic RNAs use distinc kinetic mechanisms, the most striking difference being the propensity of the hairpin ribozyme to catalyze RNA ligation. pH and divalent metal cation independence of cleavage and ligation kinetics also point to a unique catalyti mechanism for the hairpin ribozyme. The investigator proposes to develop and test a detailed model of hairpin ribozyme catalytic chemistry. To ask if the highest energy barrier to hairpin cleavage is breaking the bond between phosphorus and the 5' oxygen, the investigator will determine cleavage rate constants for a hairpin substrate in which the reactive phosphodiester has bee replaced with a 5'-bridging phosphorothioate ester. Because sulfur is a better leaving group than oxygen, sulfur substitution of the bridging 5' oxygen is expected to accelerate phosphorus-sulfur bond breaking. If this step is rate-determining, cleavage of the thioester will be faster than cleavage of th natural substrate. Because a role for metal hydroxide as the general base in hairpin catalysis has been excluded, the investigator will examine functional groups in the ribozyme for the ability to catalyze proton transfer. Heteronuclear NMR experiments will be used to identify functional groups that might exhibit shifted pK values. Enhanced sensitivity to covalent modification by electrophilic reagents may reveal a potential general base catalyst within the hairpin ribozyme RNA. Nucleotide bases that might act as general acid or base catalysts will be replaced by base analogs with altered pKa values. Chang in the pH dependence of catalytic rate constants that results from the introduction of a modified nucleotide base with a shifted pKa value would provide strong evidence for the role of that base in catalytic chemistry. The investigator will attempt to identify ground state interactions that reflect a unusual ionization state of a nucleotide base. Finally, the investigator will explore why the hairpin ribozyme is a better ligase than it is a nuclease by determining the thermodynamics parameters of the internal equilibrium between cleavage and ligation and by assessing the role of tertiary interactions in stabilizing the ribozyme-substract complex.