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: 10305610
• 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 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10305610
• 关键词: Acids; Active Sites; Address; Adopted; Architecture; Benchmarking; Biochemical; Biological; Biological Models; Biology; Catalysis; Catalytic RNA; Chemicals; 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.

摘要 核酸内切酶是一类非编码RNA，几乎影响RNA的各个方面 通过催化2 '-O-转磷酸化反应，代谢和形成细胞转录组。高 这些自裂解基序的解析结构揭示了不同的结构，并提供了物理上的 研究催化的结构基础。最常见的是，核碱基位于活性区。 准备好直接参与催化的位置。对于某些核酶，这些核碱基与 一般的酸碱催化作用，并显示参与催化相互作用。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制的理解和目前研究的重大局限性， 这些方法阻碍了对结构如何赋予催化作用的定量理解。 因为没有一个单一的核酶的活性位点相互作用被实验确定和解剖在一个完整的系统中。 全面的方式，也没有过渡国家的结构，可以说是最关键的特点， 理解催化作用，被描述。因此，理论家缺乏适当的数据来衡量 和先进的计算方法。此外，活性位点内的相似性和差异也 提出了关于这些核酶的序列结构和进化关系的问题。做 核酸内切核酶独立产生，并由于化学作用而汇聚在共同的机制上。 还是它们的突变途径交叉，使得从共同祖先进化成为可能？我们 对生物学的理解和操纵及应用生物学的能力关键在于理解催化作用， 它的进化机制，因为化学反应必须以超过自然耗散力的速度发生 允许生命系统创造秩序，维持组织，并进化。从长远来看，我们希望 对核酶催化作用的结构和进化起源的定量、预测性理解。这 本申请有两个总体目标：（1）通过VS核酶产生催化的原子图像， 结合了过渡态键合信息、质子转移的位置和程度，以及过渡态的位置和程度。 在整个三级结构的背景下，状态相互作用，以及（2）确定是否适合 VS和发夹状核酶相交的一个看似合理的进化前体的景观。完成 第一个目标以全面的方式将是任何催化剂的一个里程碑; 后一个目标将强调RNA自切割基序可以出现并建立RNA的流动性。 核酸内切核酶之间共同祖先的可能性。在我们最近高分辨率 VS核酶的结构，我们将启动新的实验策略，确定催化相互作用 使用双突变体循环，解释伴随的pKa位移，测量重原子动力学同位素 影响，并将该领域超越从结构接近性推断质子转移，以获得实际的 一般酸碱催化和相关的布朗斯台德系数的生化签名。