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Mechanisms of DNA and RNA Transactions

DNA 和 RNA 交易的机制

基本信息

批准号：
10618537
负责人：
Stewart H Shuman
金额：
$ 108.32万
依托单位：
SLOAN-KETTERING INST CAN RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-05-03 至 2028-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10618537
关键词：
Antifungal Agents Archaea Bacteria Bacteriophage T4 Biochemistry Biological Models Cell physiology Cells Cellular Stress Code Complex DNA Diphosphates Enzymes Event Family Fission Yeast Functional disorder Gene Expression Gene Expression Regulation Genetic Genetic Transcription Goals Guanosine Triphosphate Homeostasis Human Human Genome Immune response Inositol Ligase Mediating Messenger RNA Microbiology Modification Mycoses Normal Cell Nucleic Acids Nucleotides Pathway interactions Phosphotransferases Phylogenetic Analysis Physiology Proteins RNA RNA Ligase (ATP)RNA Splicing Reaction Regulation Regulon Repression Research Retrotransposition Signaling Molecule Specificity Structure System TPT1 gene Transfer RNA Virus Virus Diseases cofactor drug discovery enzyme structure fungus gene repression inorganic phosphate promoter pyrophosphatase repair enzyme repaired response structural biology tRNA Ligase tool transcription termination

项目摘要

PROJECT SUMMARY: The goals of the research proposed for the MIRA renewal are: (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 structures, mechanisms, 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 in complexes with nucleic acid and nucleotide substrates and cofactors, and endeavor to capture structural snapshots of intermediates and transition-states along the reaction pathways. (2) The structural basis for RNA recognition and strand joining by ATP-dependent 5'-PO4/3'-OH RNA ligase T4 Rnl1. Rnl1 is a tRNA repair enzyme that the T4 bacteriophage uses to evade a tRNA-damaging host response to virus infection. (3) The unique catalytic mechanism, end-specificity, and regulation of GTP-dependent 3'-PO4/5'-OH RNA ligase RtcB. The RtcB-family ligases are found in all phylogenetic domains. They are agents of diverse RNA transactions, including tRNA splicing (in metazoa and archaea), RNA repair (in bacteria), nonspliceosomal mRNA splicing (in the metazoan unfolded protein response), and the formation of chimeric RNAs in human cells that can undergo retrotransposition into the human genome. (4) Tandem transcriptional interference as a controlling factor in fission yeast phosphate homeostasis. The three S. pombe PHO regulon genes are repressed in phosphate-replete cells by transcription in cis of 5’- flanking lncRNAs that interferes with the PHO mRNA promoters. The lncRNA-mediated interference that underlies the repression of pho1 has afforded us a sensitive read-out of genetic influences on 3'- processing/termination and a powerful tool for discovery of agents and regulators of this step of the Pol2 transcription cycle. These influences include: (i) the Pol2 CTD code; (ii) numerous components of the 3'- processing/termination machinery; and (iii) metabolite control by inositol pyrophosphate 1,5-IP8, an intracellular signaling molecule. We propose to investigate the fission yeast Asp1 kinase/pyrophosphatase enzyme that determines IP8 dynamics and the cellular proteins and pathways that connect IP8 to gene expression.

项目摘要：为 MIRA 更新提出的研究目标是：(i) 了解进行核酸合成、修饰和修复的酶的机制和结构； (ii) 至阐明调节这些事件的因素。该项目整合了多种实验方法（微生物学、生物化学、结构生物学、遗传学）并将它们应用于模型系统病毒到细菌到真菌。主要主题是： (1) 真菌tRNA剪接酶Trl1(tRNA)的结构、机制和独特性连接酶）和 Tpt1（tRNA 2'-磷酸转移酶）——作为正常运行所必需的 RNA 修复系统的范例细胞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 Tpt1 与核酸、核苷酸底物和辅因子形成复合物，并努力捕获反应途径中中间体和过渡态的结构快照。 (2) ATP依赖性5'-PO4/3'-OH RNA连接酶T4识别RNA和链连接的结构基础 RNl1。 Rnl1 是一种 tRNA 修复酶，T4 噬菌体用它来逃避 tRNA 损伤的宿主反应到病毒感染。 (3) GTP依赖性3'-PO4/5'-OH RNA独特的催化机制、末端特异性和调控连接酶 RtcB。 RtcB 家族连接酶存在于所有系统发育域中。它们是多种RNA的代理交易，包括 tRNA 剪接（在后生动物和古细菌中）、RNA 修复（在细菌中）、非剪接体 mRNA 剪接（在后生动物未折叠蛋白反应中）以及人类嵌合 RNA 的形成可以逆转录转座到人类基因组中的细胞。 (4)串联转录干扰作为裂殖酵母磷酸盐稳态的控制因素。这三个粟酒裂殖酵母 PHO 调节子基因在磷酸盐充足的细胞中通过 5'- 顺式转录受到抑制干扰 PHO mRNA 启动子的侧翼 lncRNA。 lncRNA介导的干扰 pho1 抑制的基础使我们能够灵敏地读出对 3'- 的遗传影响处理/终止以及发现 Pol2 这一步的代理和调节器的强大工具转录周期。这些影响包括： (i) Pol2 CTD 代码； (ii) 3'-的众多成分加工/终止机械； (iii) 通过肌醇焦磷酸 1,5-IP8（一种细胞内药物）控制代谢信号分子。我们建议研究裂殖酵母 Asp1 激酶/焦磷酸酶确定 IP8 动态以及将 IP8 与基因表达连接的细胞蛋白和途径。