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：该MIRA提议合并并扩展了潜水员的调查线 DNA和RNA交易由四个长期的Nigms赠款支持。目标 这项研究是：（i）了解执行核酸的酶的机制和结构 合成，修改和修复； （ii）阐明调节这些事件的因素。项目 整合潜水员实验方法（微生物学，生物化学，结构生物学，遗传学）和 将它们应用于从病毒到细菌再到真菌的系统建模。主要主题是： （1）多核苷酸连接酶和mRNA的终端识别的化学机制和结构基础 通过共价酶（莱赛 - Nζ） - NMP中间体。我们将解决示例性ATP依赖性DNA连接酶的结构和封盖 将酶作为NTP和金属辅因子的步骤1 Michaelis复合体。我们将阐明 NHEJ连接酶LIGD，用于3'单核核苷酸镍和PPRNA的封盖酶。我们将运用时间 - 衰减晶体学以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，机理和独特的规范 和TPT1（TRNA 2'-磷酸转移酶） - 作为正常细胞必不可少的RNA修复系统的范例 生理学和抗真菌药物发现的有希望的靶标。我们将确定TRL1和 来自人类真菌病原体的TPT1曲霉烟曲霉和白色念珠菌的复合体 底物，辅因子和反应中间体。 （3）真核生物tRNA反登基核酸酶“核糖毒素”的机理和独特的靶标特异性（Pichia 相思毒素；拍打）物种是自称歧视的基础。我们将确定PAT的结构 及其trnagln（UUG）的底物反登起环的复合物。我们将通过 通过求解PAT·IMMPAT异二聚体的结构，pichia acaciae抗毒素IMMPAT。 （4）RNA聚合酶II（POL2）CTD代码。 POL2 CTD，由共识的串联七肽组成 序列Y1S2P3T4S5P6S7对于生存力至关重要，因为它报告了调节转录的蛋白质， 修饰染色质结构，并催化或调节mRNA封盖，剪接和聚腺苷酸化。经过 遗传操纵裂变酵母CTD，并测量对细胞生长和基因表达的影响，我们：（i） 守则的每个“字母”教育的结构活动关系； （ii）定义的字母组合 由细胞因素“读取”以及控制特定表达程序的“读取”。我们集中精力 关于CTD和转录因子PHO7在裂变酵母磷酸稳态中的作用，一种机制 在磷酸化的细胞中反映了磷酸盐的基因基因（以某种方式取决于 CTD磷酸化状态），并因磷酸盐饥饿而激活（依赖于PHO7的事件）。