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

VS 核酶：催化机制、过渡态结构和进化

基本信息

批准号：
10582360
负责人：
Joseph Anthony Piccirilli
金额：
$ 7.57万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-01-01 至 2023-11-30
项目状态：
已结题

项目摘要

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.

家长奖项目摘要核酸内切核酶代表了一类非编码 RNA，几乎影响 RNA 的各个方面通过催化 2'-O-转磷酸化反应来调节细胞的代谢和形成细胞转录组。这些自裂解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结构基础的框架最常见的是，核碱基位于活性区域。对于某些核酶来说，这些核碱基参与了直接参与催化的位点。然而，在催化相互作用方面仍存在重大差距。我们对每种核酸内切核酶的机制理解以及当前的重大局限性方法阻碍了对结构如何赋予催化作用的定量理解。因为没有任何一种核酶的活性位点相互作用经过实验鉴定和解剖。全面的方式也没有过渡国家结构，这可以说是最关键的特征对于催化的理解和表征，理论家缺乏适当的数据来进行基准测试。此外，活性位点内的相似点和差异也存在。确实提出了有关这些核酶的序列结构和进化关系的问题。核酸内切核酶独立产生，并由于化学作用而汇聚于共同机制限制还是它们的突变途径交叉，使我们的共同祖先进化成为可能？对生物学的理解以及操纵和应用生物学的能力关键取决于对催化和应用的理解它的进化机制，因为化学反应必须以超过自然耗散力的速率发生让生命系统能够创造秩序、维持组织并不断发展。从长远来看，我们希望能够发展。对核酶催化的结构和进化起源的定量、预测性理解。应用程序有两个总体目标：（1）生成 VS 核酶催化的原子图包含过渡态键合信息、质子转移的位置和范围以及过渡整体三级结构背景下的状态相互作用，以及（2）确定是否适合 VS 和发夹核酶的合理进化前体的景观相交。全面的第一个目标对于任何催化剂来说都是一个里程碑；后一个目标将强调 RNA 自切割基序的出现和建立的流动性核酸内切核酶之间存在共同祖先的可能性。 VS 核酶的结构，我们将启动新的实验策略来识别催化相互作用使用解释伴随 pKa 变化的双突变体循环，测量重原子动力学同位素效应，并使该领域超越从结构邻近推断质子转移到获得实际的质子转移一般酸碱催化的生化特征和相关的布朗斯特系数。