Structure-Activity Based Mechanistic Insights into Cleavage Chemistry by Self-Cleaving Nucleolytic Ribozymes

基于结构-活性的自裂解核酶裂解化学的机理见解

• 批准号: 10684151
• 负责人: DINSHAW J PATEL
• 金额: $ 33.63万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10684151
• 关键词: 3-Dimensional; Acids; Active Sites; Address; Architecture; Binding; Binding Sites; Biochemical; Biogenesis; Biological; Catalysis; Catalytic RNA; Cations; Chemicals; Chemistry; Collaborations; Conserved Sequence; Crystallization; Curiosities; Dependence; Divalent Cations; Elements; Enzymes; Event; Evolution; Formulation; Gene Expression Regulation; Genes; Genomics; Goals; Hepatitis Delta Virus; Human; Hydration status; Hydrogen Bonding; Hydrophobic Interactions; Introns; Life; Ligation; Literature; Measurement; Measures; Mediating; Messenger RNA; Modification; Molecular Conformation; Monitor; Oxygen; Peptides; Peptidyltransferase; Play; Positioning Attribute; Publishing; RNA; RNA Processing; RNA Splicing; RNase P; Reporting; Ribosomes; Role; Screening Result; Sister; Site; Specificity; Spliceosomes; Structure; Structure-Activity Relationship; Temperature; Time; Untranslated RNA; Untranslated Regions; Vanadates; Vertebral column; Visit; analog; base; comparative; follow-up; functional group; genome integrity; genome wide screen; inorganic phosphate; insight; interest; macromolecule; mutant; phosphodiester; prebiotics; protonation; varkud satellite ribozyme

ABSTRACT Our mechanistic understanding of small (<150 nt) self-cleaving nucleolytic ribozymes that primarily use general- acid base catalysis involving attack of the 2’-OH on the adjacent scissile phosphate to site-specifically cleave the intervening phosphodiester backbone has initially focused on hammerhead, hairpin, glmS, hepatitis delta virus and Varkud Satellite ribozymes, and more recently on twister (Twr), twister-sister (TSr), pistol (Psr) and hatchet (Htr) ribozymes. Our overall goal as reflected from our structure-function studies of the Twr, Tsr, Psr and Htr ribozymes is to expand on our current understanding of the catalytic versatility of RNA with the emphasis on the contributions of active site organization, geometric constraints, activation of the 2’-OH nucleophile, the role of transition state stabilization, protonation of the 5’-oxygen leaving group and the potential of Mg2+ cations in mediating catalysis. One of the challenges in the field relates to whether self-cleaving ribozymes use a common or diverse set of mechanisms, and the extent to which hydrated divalent cations catalyze cleavage chemistry. We have an ongoing collaboration with the Ronald Micura lab (Innsbruck) to study catalytic mechanisms of self- cleaving ribozymes by solving crystal structures of precatalytic, transition and product states in our lab, followed by systematic multi-faceted studies of structure-guided selective catalytic mutants and analogs, as well as pKa measurements of catalytic residues, and pH and temperature-dependence of catalytic rates by the Micura lab. Aim 1: A recent biochemical genome-wide screen resulted in the identification of the naturally occurring self- cleaving hovlinc ribozyme in humans. The sequence of the 168-nt hovlinc ribozyme and its 83-nt minimal functional counterpart contained two pseudoknots with one of them embedded in the cleavage site. The cleavage rate was shown to increase with pH and its inverse correlation with the pKa of divalent cations suggested the catalytic participation of a hydrated divalent cation in cleavage chemistry. We propose to crystallize and determine the structures of the precatalytic, vanadate transition-state mimic and product states of the minimal functional hovlinc ribozyme and follow up with systematic functional studies with the Micura lab of structure- guided modifications and rate measurements towards elucidation of its catalytic cleavage mechanism. Aim 2. This Aim revisits structure-activity relationships of the Twr and TSr ribozymes to resolve discrepancies in the published precatalytic structures and resulting mechanistic insights reported in the literature. The splayed- apart orientation of bases at the cleavage site in a four-way junctional TSr ribozyme by our group contrasts with the stacked bases at the cleavage site in a three-way junctional TSr from the David Lilley lab. We propose to characterize vanadate transition-state mimics of the Twr and TSr ribozymes to resolve the existing discrepancies and mechanistically evaluate the proposed role of catalytic bases and hydrated divalent cations in mediating cleavage chemistry. These studies should elucidate whether Twr and TSr ribozymes, that contain common but topologically distinct conserved sequence elements, use similar or distinct mechanisms for cleavage chemistry.

摘要 我们对小的（<150 nt）自切割溶核核酶的机制的理解，主要是使用一般的- 一种酸碱催化作用，涉及2 '-OH对相邻易裂磷酸盐的攻击，以位点特异性地裂解 介入的磷酸二酯骨架最初集中在锤头状、发夹状、glmS、丁型肝炎 病毒和Varkud卫星核酶，以及最近关于扭转酶（Twr），扭转酶-姐妹酶（TSr），手枪酶（Psr）和 斧形（Htr）核酶。我们的总体目标反映在我们对Twr、Tsr、Psr和 Htr核酶是扩大我们目前的理解，催化的多功能性的RNA与重点 活性中心组织的贡献，几何约束，2 '-OH亲核试剂的活化， 的过渡态稳定，质子化的5 '-氧离去基团和潜在的镁离子在 介导催化作用。该领域的挑战之一涉及自切割核酶是否使用共同的 或多种机制，以及水合二价阳离子催化裂解化学的程度。 我们与罗纳德Micura实验室（因斯布鲁克）正在进行合作，研究自催化机制。 本实验室通过解催化前态、过渡态和产物态的晶体结构来切割核酶， 通过对结构导向的选择性催化突变体和类似物以及pKa的系统多方面研究， Micura实验室对催化残留物的测量，以及催化速率的pH和温度依赖性。 目的1：最近的一项生物化学全基因组筛选鉴定了自然发生的自体免疫缺陷病毒。 切割人类的hovlinc核酶。Hovlinc核酶的168-nt和83-nt最小核苷酸序列 功能对应物含有两个假结，其中一个嵌入切割位点。裂解 速率随pH值的增加而增加，其与二价阳离子的pKa呈负相关，表明 水合二价阳离子在裂解化学中的催化参与。我们建议结晶和 确定最小催化剂的预催化、钒酸盐过渡态模拟物和产物态的结构， 功能性hovlinc核酶，并与Micura结构实验室进行系统的功能研究- 引导修饰和速率测量，以阐明其催化裂解机制。 目标二。本研究的目的是重新审视Twr和TSr核酶的结构-活性关系，以解决差异 在已发表的预催化结构和由此产生的机械见解在文献中报道。张开的- 我们的小组在四向连接TSr核酶中切割位点处碱基的分离方向与 大卫利实验室的三向连接TSr中切割位点的堆叠碱基。我们建议 表征Twr和TSr核酶的钒酸过渡态模拟物，以解决现有的差异 并从机理上评估催化碱和水合二价阳离子在介导 卵裂化学这些研究应该阐明是否Twr和TSr核酶，含有共同的，但 拓扑上不同的保守序列元件使用相似或不同的切割化学机制。