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

• 批准号: 10061618
• 负责人: Joseph Anthony Piccirilli
• 金额: $ 32.31万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-01-01 至 2022-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10061618
• 关键词: Acids; Active Sites; Address; Adopted; Architecture; Benchmarking; Biochemical; Biological; Biological Models; Biology; Catalysis; Catalytic RNA; Chemicals; Cleaved cell; Data; Disease; Evolution; Goals; Health; Isotopes; Kinetics; Laboratories; Location; Measurement; Measures; Mechanics; Modeling; Mutagenesis; Mutation; Organism; Outcome; Oxygen; Pathway interactions; Play; Process; Protons; RNA; RNA Folding; RNA metabolism; Reaction; Resolution; Role; Shapes; Structure; Testing; Transferase; Untranslated RNA; Variant; Work; base; catalyst; chemical reaction; fitness; fluidity; hairpin ribozyme; iterative design; mutant; nucleobase; quantum; simulation; transcriptome; varkud satellite ribozyme

ABSTRACT 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.

抽象的 核核核酶代表一类非编码RNAT影响RNA的几乎存在 代谢和通过2'-O-转磷酸反应的催化作用 该链接图案的分辨率结构揭示了不同的架构并提供物理 框架研究催化的结构基础。 站点直接参与催化作用。 一般酸碱催化并证明参与催化相互作用。 我们机械理解的野生核核酶核酶和电流的显着局限性 方法阻碍了对结构如何进行催化的定量理解的方式。 对于任何单个核酶没有实验鉴定并在 全面的方式没有过渡状态结构，Arguaby是最关键的特征 理解催化剂，因此，理论家缺乏适当的数据 并推进计算方法。 提出有关这些核心的序列结构和进化关系的问题。 内核酸核酶会持悬念，并根据化学物质汇聚 限制还是它们的突变途径相交，使我们的养老金进化？ 对操纵和应用生物学操纵和运用生物学的理解和能力在理解催化和 它的进化机制，因为必须以超过natpace natpatipativative力的速率发生化学反应 为了使生活系统折痕，维护组织和发展。 对核酶催化的结构和进化起源的定量，预测的理解。 应用程序有两个总体目标：（1）生成与核酶的催化作用的原子图。 结合过渡状态键合信息，质子转移的位置和范围以及过渡 在整个第三级结构的背景下的状态相互作用，（2）确定适合度是否 VS和发夹核酶的合理进化前体的景观 以全面的方式，第一个目标是任何催化剂的里程碑； 后一个目标将强调RNA自我切割图案可以建立脚趾的流动性 在我们最近的高分辨率上建立的核心核酶中的共同血统的可能性 与核酶的结构，我们将确定鉴定催化相互作用的新实验策略 使用伴随PKA偏移的双重突变循环，测量重原子动力学 效果，并将领域超越蛋白质超越蛋白 生化特征通用一般酸碱催化和相关的Brønsted系数。