CATALYTIC MECHANISM OF THE HAIRPIN RIBOZYME

描述：这项工作的长期目标是阐明 由小核糖核酸酶催化。分子间发夹的详细分析 锤头状核酶反应表明，这两个催化RNA使用 动力学机制，最显著的区别是 发夹状核酶催化RNA连接的倾向。PH值和 二价金属阳离子对切割和连接动力学的独立性 指出发夹状核酶的独特催化机制。这个 研究人员建议开发和测试发夹的详细模型 核酶催化化学。要问最高的能量屏障是否 发夹裂解正在破坏磷和5‘氧之间的键， 研究人员将确定发夹的切割速率常数 反应性磷酸二酯已被取代的底物 5‘-桥联硫代磷酸酯。因为硫磺是更好的离开 与氧相比，5‘桥氧的硫取代是可望的 以加速磷硫键的断裂。如果此步骤是 速率决定，硫酯的裂解速度将快于硫酯的裂解 这是天然的底物。因为金属氢氧化物作为将军的角色 发夹催化中的碱基已被排除，调查者将检查 核酶中催化质子能力的官能团 调职。异核核磁共振实验将被用来鉴定功能 可能显示PK值发生变化的组。更高的敏感度 亲电试剂的共价修饰可能揭示出一种潜在的 发夹状核酶RNA中的通用碱基催化剂。核苷酸碱基 可能作为普通酸或碱催化剂的催化剂将被碱取代 与pKa值改变的类似物。催化剂的pH依赖性中的Chang 由引入修饰的核苷酸所产生的速率常数 PKa值发生变化的碱基将提供强有力的证据证明 这是催化化学的基础。调查员将试图确定 基态相互作用反映了一种不寻常的电离态 核苷酸碱基。最后，调查人员将探索为什么发夹 核酶是一种比核酸酶更好的连接酶，通过确定 解理和解理之间内部平衡的热力学参数 通过评估三级相互作用在稳定中的作用 核酶-底物复合体。

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.