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：这个MIRA提案巩固和扩展了对基本的调查的不同路线 迄今为止，DNA和RNA交易得到了四个长期的NIGMS赠款的支持。的目标 本研究的目的是：（1）了解核酸酶的作用机制和结构， 合成，修饰和修复;和（ii）阐明调节这些事件的因素。项目 整合不同的实验方法（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 加帽酶，其催化核苷酸基通过共价酶-（赖氨酰- NMP）-NMP中间体。我们将解决典型的ATP依赖性DNA连接酶和加帽的结构 酶作为它们的步骤1与NTP和金属辅因子的米氏络合物。我们将阐明 NHEJ连接酶LigD用于3 '-单核糖核苷酸切口，加帽酶用于ppRNA。我们会花时间- lapse晶体学来探测金属在通过连接酶的磷酸二酯合成中的作用。 (2)真菌tRNA剪接酶Trl 1（tRNA连接酶）的结构、机制和特异性 和Tpt 1（tRNA 2 ′-磷酸转移酶）-作为正常细胞必需RNA修复系统的范例 生理学和抗真菌药物发现的有前途的目标。我们将确定Trl 1的结构， 来自人类真菌病原体烟曲霉和白色念珠菌的Tpt 1与 底物、辅因子和反应中间体。 (3)真核生物tRNA反密码子核酸酶“核糖毒素”（毕赤酵母）的作用机制和特异性靶点 金合欢毒素（Acaciae toxin，PaT），是物种自我-非自我辨别的基础。我们将确定的结构， 与其底物tRNAGln（UUG）的反密码子环复合。我们将阐明保护性免疫的基础， 通过解析PaT·ImmPaT异源二聚体的结构， (4)RNA聚合酶II（Pol 2）CTD代码。Pol 2 CTD，由共有的串联七肽组成 序列Y1 S2 P3 T4 S5 P6 S7是生存力所必需的，因为它募集调节转录的蛋白质， 修饰染色质结构，并催化或调节mRNA加帽、剪接和聚腺苷酸化。通过 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） 推导出代码中每个“字母”的结构-活性关系;以及（ii）定义的字母组合， 包括由细胞因子“读取”的“字”，其控制特定的表达程序。我们专注 在这里，CTD和转录因子Pho 7在裂殖酵母磷酸盐稳态中的作用， 由此磷酸盐获取基因在磷酸盐充足的细胞中被抑制（以依赖于 CTD磷酸化状态），并响应磷酸盐饥饿而激活（依赖于Pho 7的事件）。