The VS Ribozyme: Catalytic Mechanism, Transition State Structure, and Evolution

来自家长奖的项目摘要 内切核酶代表一类非编码的RNA，几乎影响到RNA的各个方面 通过催化2‘-O-转磷酸化反应代谢和塑造细胞转录本。高 这些自切割基序的解析结构揭示了不同的结构，并提供了物理 研究催化的结构基础的框架。最常见的情况是，碱基位于活跃的 准备直接参与催化的站点。对于某些核酶来说，这些碱基与 一般的酸碱催化和表现为参与催化相互作用。然而，在以下方面存在重大差距 我们对每一种核酸内切核酶的机理理解和目前存在的重大限制 方法阻碍了对结构如何给予催化的定量理解的发展。 对于任何单一的核酶来说，活性部位的相互作用都没有在实验中被鉴定和解剖 过渡状态结构也不是全面的方式，可以说是 了解催化作用，对其进行了表征。因此，理论家缺乏适当的数据来作为基准。 和先进的计算方法。此外，活跃站点内部的相似和差异也 提出关于这些核酶的序列结构和进化关系的问题。做 核酸内切核酶由于化学作用而独立产生和聚集在共同的机制上 限制或它们的突变路径是否相交，使从共同祖先进化成为可能？我们的 对生物学的理解和操纵和应用的能力在很大程度上取决于对催化和 它的进化机制，因为化学反应的发生速度必须超过自然耗散力 使有生命的系统能够创造秩序、维持组织和进化。从长远来看，我们希望发展 对核酶催化的结构和进化起源的定量、预测性理解。这 应用程序有两个总体目标：(1)生成VS核酶催化的原子图像， 结合了过渡态键合信息、质子转移的位置和范围以及过渡 在整体三级结构背景下的状态交互作用，以及(2)确定是否适合 VS和发夹状核酶的一种看似合理的进化前体的景观相交。完成 第一个全面的目标对任何催化剂来说都是一个里程碑；实现 后一个目标将强调RNA自切割基序的流动性，通过它可以出现并建立 核酸内切核酶的共同祖先的可能性。在我们最近高分辨率的基础上 结构，我们将启动新的实验策略来识别催化相互作用 使用双突变循环来解释伴随的pKA移动，测量重原子动力学同位素 效应，并使场超越推断质子转移，从结构接近转移到获得实际的 一般酸碱催化的生物化学特征和相关的Brnsted系数。

PROJECT ABSTRACT from the Parent Award Endonucleolytic ribozymes represent a class of noncoding RNAs that influence nearly every aspect of RNA metabolism and shape cellular transcriptomes through catalysis of 2'-O-transphosphorylation reactions. High resolution structures of these self-cleavage motifs reveal distinct architectures and provide physical frameworks to investigate the structural basis of catalysis. Most commonly, nucleobases reside at the active site poised to engage directly in catalysis. For some ribozymes these nucleobases have been implicated in general acid base catalysis and shown to engage in catalytic interactions. Nevertheless, major gaps exist in our mechanistic understanding for every endonucleolytic ribozyme and significant limitations in current approaches stand in the way of developing a quantitative understanding for how structure imparts catalysis. For no single ribozyme have the active site interactions been experimentally identified and dissected in a comprehensive manner nor has the transition state structure, arguably the most critical feature in understanding catalysis, been characterized. Consequently, theoreticians lack appropriate data to benchmark and advance computational approaches. Moreover, similarities and differences within the active sites also raise questions about the sequence-structure and evolutionary relationships of these ribozymes. Did endonucleolytic ribozymes arise independently and converge upon common mechanisms due to chemical constraints or do their mutational pathways intersect, making evolution from a common ancestor possible? Our understanding of and ability to manipulate and apply biology hinges critically upon understanding catalysis and its mechanisms of evolution, as chemical reactions must occur at rates that outpace natural dissipative forces to allow living systems to create order, maintain organization, and evolve. In the long term, we hope to develop a quantitative, predictive understanding of the structural and evolutionary origins of ribozyme catalysis. This application has two overall goals: (1) to generate an atomistic picture of catalysis by the VS ribozyme that incorporates transition state bonding information, locations and extents of proton transfer, and transition state interactions in the context of the overall tertiary structure, and (2) to determine whether the fitness landscapes of a plausible evolutionary precursors of the VS and hairpin ribozymes intersect. Accomplishing the first goal in a comprehensive manner would represent a milestone for any catalyst; accomplishing the latter goal would underscore the fluidity by which RNA self-cleavage motifs can emerge and establish the possibility of common ancestry among endonucleolytic ribozymes. Building upon our recent high-resolution structure of the VS ribozyme, we will initiate new experimental strategies that identify catalytic interactions using double mutant cycles that account for concomitant pKa shifts, measure heavy atom kinetic isotope effects, and move the field beyond inferring proton transfer from structural proximity to obtaining actual biochemical signatures for general acid-base catalysis and associated BrØnsted coefficients.