RNA Modification Enzymes: Methyltransferase/PSI-synthase

RNA 修饰酶：甲基转移酶/PSI 合酶

基本信息

批准号：
7730115
负责人：
Robert M Stroud
金额：
$ 32.45万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-05-01 至 2013-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7730115
关键词：
2-Aminopurine Abbreviations Active Sites Adenine Anemia Anti-HIV Agents Anti-HIV Therapy Anticodon Bacterial RNA Base Pairing Binding Biochemical Reaction Biological Availability Catalysis Catalytic Domain Cells Code Complex Crystallization Defect Disease Drug Delivery Systems Environment Enzymes Escherichia coli Family Fluorescence Genome HIV Health Higher Order Chromatin Structure Human Human Biology Hydrogen Bonding Individual Kinetics Knowledge Label Lead Length Libraries Life Lysine-Specific tRNA Measures Methyltransferase Modeling Modification Monitor Mutagenesis Mutate Mutation Nature Nuclear Receptors Nucleotides Pathway interactions Play Positioning Attribute Proteins Pseudouridine RNA RNA Folding RNA Interference RNA Sequences Reaction Receptor Signaling Regulation Relative (related person)Reporting Reverse Transcription Ribosomal RNA Ribosomes Role S-Adenosylmethionine Screening procedure Sideroblastic Anemia Site Specificity Staging Structure Substrate Specificity Testing Therapeutic Intervention Thymine Time Transfer RNA Translating Upper arm Uracil Uridine Work base combat design enzyme structure flexibility human disease improved inhibitor/antagonist member molecular dynamics mutant prevent public health relevance research study stem steroid receptor RNA activator stopped-flow fluorescence tRNA Methyltransferases

项目摘要

DESCRIPTION (provided by applicant): In all kingdoms of life RNAs are modified both during and after synthesis, thereby altering the stability, higher-order structure, and activity of RNA through changes in base stacking and hydrogen bonding. These generally conserved modifications are located at key functional regions and are required for optimal RNA function. Approximately 0.8% of the coding capacity of the E. coli cell is dedicated to enzymes that modify RNA with unique or limited multisite specificity. The aim of this application is to determine the basis for selectivity and catalysis in two of the most abundant families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases) and pseudouridine synthases (synthases). Our reported structures of two m5U MTases have yielded a model for specific recognition in which the RNA substrate is refolded onto the enzyme and a base is flipped out into the active site. This work proposes to test, refine and elaborate that model in kinetic and structural terms. Our to-date and proposed studies of five sub-classes of bacterial (synthases that modify stem loops of tRNAs (TruA and TruB), a stem loop (RluD), and a helix (RluB and RluF)) rRNA is yielding models for site specificity and regional specificity that will serve as paradigms for synthases that play roles in human health. The field will thus be advanced into regulation in human biology with our proposed work on human Pus1 and Pus3, two enzymes that regulate nuclear receptor signaling by pseudouridylating an RNA activator of nuclear receptors (steroid receptor RNA activator, SRA). Defects in these enzymes cause sideroblastic anemia. The strategy for all enzyme studies remains to express key proteins, to define the nature of the substrate as a complete tRNA or smaller stem-loop, and then to determine crystal structures of enzyme-RNA complexes at critical points close to the transition state. Mutations in protein and RNA are used to determine the mechanisms by which the individual target base is brought into the catalytic site. Mechanism is also probed through measures of the rates of individual steps including base-flipping, kcat/Km and Kd determinations. RNA structural changes during the reactions will be monitored through time-resolve fluorescence changes of 2-aminopurine labeled substrates. Targeted molecular dynamics is used to guide experiments. Finally, we seek to determine the structure of a human tRNA adenine MTase as a potential new anti-HIV drug target. It generates an essential modification that controls reverse transcription of HIV RNA. The expression and purification of this two subunit AdoMet dependent RNA methylase is to lead to a structure, and to a screening approach to specific inhibitor discovery. PUBLIC HEALTH RELEVANCE: The aim is to determine the basis for action of two of the most abundant families of RNA modifying enzymes: 5-methyl uracil (m5U) methyltransferases (MTases), and pseudouridine synthases (synthases), and to elucidate the roles of two human synthases and one human RNA MTase with importance in human disease. The importance of synthase activity is underlined by disorders such as sideroplastic anemia. The RNA MTase is a potential new drug target for anti-HIV therapy.

描述（由申请人提供）：在合成期间和之后，在生命的所有王国中，RNA都经过修改，从而改变了RNA的稳定性，高阶结构以及RNA的活性，通过基础堆叠和氢键的变化来改变RNA的活性。这些通常保守的修改位于关键功能区域，是最佳RNA功能所必需的。大肠杆菌细胞的编码能力的大约0.8％专用于用独特或有限的多站点特异性修饰RNA的酶。该应用的目的是确定两个最丰富的RNA修饰酶家族的选择性和催化的基础：5-甲基尿嘧啶（M5U）甲基转移酶（MTases）和假喹啉合酶（合成酶）。我们报道的两个M5U MTase的结构产生了一个特定识别模型，其中RNA底物被重新折叠成酶，并将碱倒入活性位点。这项工作建议用动力学和结构术语进行测试，完善和阐述该模型。我们对五个子类细菌（修饰TRNA（TRUA和TRUB）茎环的合成酶，茎环（RLUD）以及Helix（RLUB和RLUF）RRNA的五个子类的研究和拟议的研究是为地点特异性和区域特异性的产生模型，这些模型将作为人类健康中的合成酶的态度。因此，通过我们提出的关于人PUS1和PUS3的工作，该领域将在人类生物学的调节中，这是两个酶，这些酶通过假硫岛通过核受体（类固醇受体RNA活化剂SRA）来调节核受体信号传导。这些酶的缺陷会引起副细胞贫血。所有酶研究的策略仍然是表达关键蛋白，将底物的性质定义为完整的tRNA或较小的茎环，然后确定接近过渡状态的临界点的酶-RNA复合物的晶体结构。蛋白质和RNA中的突变用于确定将单个靶基碱带入催化位点的机制。还通过测量包括基本底面，KCAT/KM和KD确定的单个步骤速率来探测机制。在反应过程中的RNA结构变化将通过2-氨基嘌呤标记的底物的荧光变化来监测。靶向分子动力学用于指导实验。最后，我们试图确定人tRNA腺嘌呤MTase的结构，作为潜在的新抗HIV药物靶标。它产生了控制HIV RNA的逆转录的基本修饰。这两个亚基ADOMET依赖RNA甲基酶的表达和纯化是导致结构，并筛选特定抑制剂发现的方法。公共卫生相关性：目的是确定两个最丰富的RNA修饰酶的作用的基础：5-甲基尿素（M5U）甲基转移酶（MTases）（MTases）和假氨基氨基氨基合酶（合成酶），以及阐明两种人类合成酶和一种具有人类RNA MTase的作用。合成酶活性的重要性是由诸如毒状贫血等疾病强调的。 RNA MTase是抗HIV治疗的潜在新药物。