Mechanisms of DNA and RNA transactions

DNA 和 RNA 交换的机制

• 批准号: 9922973
• 负责人: Stewart H Shuman
• 金额: $ 107.76万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-03 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9922973
• 关键词: Anticodon; Antifungal Agents; Antineoplastic Agents; Antiviral Agents; Aspergillosis; Aspergillus fumigatus; Award; Bacteria; Biochemistry; Biological Models; Candida albicans; Candidiasis; Cell physiology; Cells; Chemicals; Chromatin Structure; Cleaved cell; Code; Complex; Consensus Sequence; Crystallography; DNA; DNA Ligases; DNA Repair Enzymes; Defect; Discrimination; Enzymes; Event; Fission Yeast; Functional disorder; Gene Expression; Genes; Genetic; Genetic Diseases; Genetic Transcription; Glutamine-Specific tRNA; Goals; Grant; Homeostasis; Human; Human Genetics; Immune; Immunity; Letters; Ligase; Malignant Neoplasms; Metals; Microbiology; Modification; Mycoses; National Institute of General Medical Sciences; Nonhomologous DNA End Joining; Normal Cell; Nucleic Acids; Pathway interactions; Phase; Phosphotransferases; Physiology; Pichia; Polyadenylation; Polymerase; Polynucleotide Ligases; Proteins; RNA; RNA Polymerase II; RNA Splicing; Reaction; Research; Role; Specificity; Starvation; Structure; System; TPT1 gene; Time; Toxin; Transact; Transfer RNA; Virus; anticodon nuclease; antitoxin; cell growth; cofactor; drug discovery; enzyme structure; fungus; genetic manipulation; inorganic phosphate; mRNA capping; mRNA guanylyltransferase; pathogenic fungus; phosphodiester; programs; recruit; repaired; response; structural biology; tRNA Ligase; transcription factor; virtual

PROJECT SUMMARY: This MIRA proposal consolidates and extends diverse lines of inquiry into fundamental DNA and RNA transactions that were heretofore supported by four longstanding NIGMS grants. The goal of this research is: (i) to understand the mechanisms and structures of enzymes that perform nucleic acid synthesis, modification, and repair; and (ii) to elucidate factors that regulate these events. The project integrates diverse experimental approaches (microbiology, biochemistry, structural biology, genetics) and applies them to model systems ranging from viruses to bacteria to fungi. The principal themes are: (1) The chemical mechanism and structural basis for end recognition by polynucleotide ligases and mRNA capping enzymes that catalyze nucleotidyl transfer to 5' phosphorylated ends via a covalent enzyme-(lysyl- Nζ)–NMP intermediate. We will solve structures of exemplary ATP-dependent DNA ligases and capping enzymes as their step 1 Michaelis complexes with NTP and metal cofactors. We will clarify the specificity of the NHEJ ligase LigD for a 3'-monoribonucleotide nick and of capping enzyme for ppRNA. We will employ time- lapse crystallography to probe the role of metals in phosphodiester synthesis by ligases. (2) The structure, mechanism, and distinctive specificities of fungal tRNA splicing enzymes Trl1 (tRNA ligase) and Tpt1 (tRNA 2'-phosphotransferase) – as paradigms of an RNA repair system essential for normal cell physiology and as promising targets for anti-fungal drug discovery. We will determine structures of Trl1 and Tpt1 from the human fungal pathogens Aspergillus fumigatus and Candida albicans in complexes with substrates, cofactors, and reaction intermediates. (3) The mechanism and distinctive target specificity of a eukaryal tRNA anticodon nuclease “ribotoxin” (Pichia acaciae toxin; PaT) that underlies species self-nonself discrimination. We will determine the structure of PaT in complex with its substrate anticodon loop of tRNAGln(UUG). We will illuminate the basis for protective immunity by the Pichia acaciae antitoxin ImmPaT by solving the structure of a PaT·ImmPaT heterodimer. (4) The RNA polymerase II (Pol2) CTD code. The Pol2 CTD, consisting of tandem heptapeptides of consensus sequence Y1S2P3T4S5P6S7, is essential for viability because it recruits proteins that regulate transcription, modify chromatin structure, and catalyze or regulate mRNA capping, splicing, and polyadenylation. By genetically manipulating the fission yeast CTD, and gauging effects on cell growth and gene expression, we: (i) educed structure-activity relations for each “letter” of the code; and (ii) defined combinations of letters that comprise “words” that are “read” by cellular factors, and which govern specific expression programs. We focus here on the roles of CTD and transcription factor Pho7 in fission yeast phosphate homeostasis, a mechanism whereby phosphate-acquisition genes are repressed in phosphate-replete cells (in a manner dependent on CTD phospho-status), and activated in response to phosphate starvation (an event dependent on Pho7).

项目摘要：MIRA 提案巩固并扩展了对基本原理的不同研究方向 迄今为止，DNA 和 RNA 交易均由 NIGMS 的四项长期拨款支持。目标是 这项研究是：（i）了解执行核酸的酶的机制和结构 合成、修饰和修复； (ii) 阐明调节这些事件的因素。项目 整合了不同的实验方法（微生物学、生物化学、结构生物学、遗传学） 将它们应用于从病毒到细菌到真菌的模型系统。主要主题是： (1) 多核苷酸连接酶和mRNA末端识别的化学机制和结构基础 通过共价酶-（赖氨酰-）催化核苷酸转移至 5' 磷酸化末端的加帽酶 Nz)–NMP中间体。我们将解决示例性 ATP 依赖性 DNA 连接酶和加帽的结构 酶作为其步骤 1 米氏复合物与 NTP 和金属辅助因子。我们将澄清该问题的特殊性 用于 3'-单核糖核苷酸切口的 NHEJ 连接酶 LigD 和用于 ppRNA 的加帽酶。我们将利用时间- 延时晶体学探测金属在连接酶合成磷酸二酯中的作用。 (2) 真菌tRNA剪接酶Trl1（tRNA连接酶）的结构、机制和独特性 和 Tpt1（tRNA 2'-磷酸转移酶）——作为正常细胞必需的 RNA 修复系统的范例 生理学和作为抗真菌药物发现的有希望的目标。我们将确定 Trl1 和 来自人类真菌病原体烟曲霉和白色念珠菌的 Tpt1 复合物 底物、辅因子和反应中间体。 （3）真核tRNA反密码子核酸酶“核毒素”（毕赤酵母）的作用机制和独特的靶点特异性 金合欢毒素； PaT）是物种自我-非自我歧视的基础。我们将确定PaT的结构 与其底物反密码子环 tRNAGln(UUG) 形成复合物。我们将通过以下方式阐明保护性免疫的基础： 通过解析PaT·ImmPaT异二聚体的结构，得到金合欢毕赤酵母抗毒素ImmPaT。 (4) RNA聚合酶II (Pol2) CTD代码。 Pol2 CTD，由一致的串联七肽组成 序列 Y1S2P3T4S5P6S7 对于生存能力至关重要，因为它招募调节转录的蛋白质， 修饰染色质结构，并催化或调节 mRNA 加帽、剪接和聚腺苷酸化。经过 通过基因操作裂殖酵母 CTD，并测量对细胞生长和基因表达的影响，我们：(i) 导出代码每个“字母”的结构-活动关系； (ii) 定义的字母组合 包含由细胞因子“读取”的“单词”，并控制特定的表达程序。我们专注 CTD 和转录因子 Pho7 在裂殖酵母磷酸盐稳态中的作用，这是一种机制 由此磷酸盐获取基因在磷酸盐充足的细胞中受到抑制（以依赖于磷酸盐的方式） CTD 磷酸状态），并响应磷酸盐饥饿（依赖于 Pho7 的事件）而激活。