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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.

项目概述：MIRA更新的研究目标是：（i）了解进行核酸合成、修饰和修复的酶的机制和结构;以及（ii）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），并将其应用于模型系统，病毒到细菌再到真菌。主要主题是： (1)真菌tRNA剪接酶Trl 1（tRNA）的结构、机制和独特的特异性连接酶）和Tpt 1（tRNA 2 '-磷酸转移酶）-作为正常细胞所必需的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前途的目标。我们将确定Trl 1的结构， Tpt 1与核酸、核苷酸底物和辅因子形成复合物，并奋进捕获沿着反应途径的中间体和过渡态的结构快照。 (2)ATP依赖的5 '-PO 4/3'-OH RNA连接酶T4识别RNA和连接RNA链的结构基础 Rnl 1。Rnl 1是一种tRNA修复酶，T4噬菌体使用它来逃避tRNA破坏性宿主反应病毒感染。 (3)GTP依赖的3 '-PO 4/5'-OH RNA的独特催化机制、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。它们是不同RNA的代理人包括tRNA剪接（后生动物和古生菌），RNA修复（细菌），非剪接体 mRNA剪接（在后生动物未折叠蛋白反应中）和人类嵌合RNA的形成可以进行逆转录转座进入人类基因组的细胞。 (4)串联转录干扰作为裂殖酵母磷酸盐稳态的控制因子。的三个S粟酒裂殖酵母PHO调节子基因在充满磷酸盐的细胞中通过5 '- 干扰PHO mRNA启动子的侧翼lncRNA。lncRNA介导的干扰，是pho 1抑制的基础，这为我们提供了一个敏感的关于3 '- 处理/终止和一个强大的工具，用于发现代理和监管机构的这一步骤的Pol 2 转录周期这些影响包括：（i）Pol 2 CTD代码;（ii）3 '- 加工/终止机制;和（iii）代谢物控制肌醇焦磷酸1，5-IP 8，细胞内信号分子我们建议研究裂殖酵母Asp 1激酶/焦磷酸酶，决定了IP 8的动力学和细胞蛋白质以及将IP 8与基因表达连接起来的途径。