Molecular Mechanism and Biological Function of 3'-5' Nucleotide Addition

3-5核苷酸添加的分子机制和生物学功能

• 批准号: 7986833
• 负责人: Jane Elizabeth Jackman
• 金额: $ 27.72万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-15 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7986833
• 关键词: Active Sites; Affect; Antifungal Agents; Antiparasitic Agents; Archaea; Bacteria; Base Pairing; Biochemical; Biological; Biological Assay; Biological Process; Biology; Catalysis; Cells; DNA; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Defect; Development; Diabetic Nephropathy; Enzyme Kinetics; Enzymes; Eukaryota; Exhibits; Family; Family member; Genetic; Genome; Health; Histidine-Specific tRNA; Homologous Gene; Human; Investigation; Kinetics; Lead; Life; Link; Mitochondria; Molecular; Nucleic Acids; Nucleotides; Organism; Pathology; Pathway interactions; Phenotype; Plasmodium falciparum; Property; Proteins; Protozoa; Reaction; Role; Small RNA; Techniques; Testing; Transfer RNA; Trichomonas vaginalis; Variant; Yeasts; abstracting; base; biological systems; enzyme activity; guanylyltransferase; human disease; in vivo; insight; member; novel; overexpression; pathogen; prevent; protein function; public health relevance; repaired

DESCRIPTION (provided by applicant): Project Summary/Abstract: The tRNAHis guanylyltransferase (Thg1) is absolutely essential in yeast, and likely throughout all eukaryotes, due to the universal requirement for G-1 on tRNAHis in all eukaryotes in which it has been investigated. Thg1 adds G-1 to tRNAHis via an unusual non-templated 3'-5' nucleotide addition reaction, by an unknown catalytic mechanism that cannot be predicted based on similarity to known enzymes, and thus is likely to employ a novel catalytic mechanism. Moreover, we have recently demonstrated that all Thg1 family members catalyze a template-dependent 3'-5' addition reaction with various substrates, and that this activity is used for a form of G-1 addition in archaea, as well as for an unusual tRNA editing reaction in protozoa. These demonstrated roles for templated 3'-5' addition greatly expand the scope of catalytic activities exhibited by Thg1 family members. Nonetheless, the presence of Thg1 homologs in archaea and bacteria that do not require enzymatic G-1 addition to tRNAHis and unexplained Thg1-related phenotypes in yeast and human cells suggest that additional roles for 3'-5' addition are yet to be uncovered. This application proposes the use of kinetic, genetic, biochemical and structural techniques to investigate the molecular mechanisms and biological functions of both non-templated and templated 3'-5' addition reactions catalyzed by diverse Thg1 family members. These results will provide insight into catalysis of a novel and apparently widespread, but largely unexplored, reaction in biology, and will enable further investigation into alternative functions for 3'-5' nucleotide addition in biological systems. PUBLIC HEALTH RELEVANCE: Project Narrative Investigation of the unusual 3'-5' nucleotide addition reactions catalyzed by Thg1 family members is of importance to human health, due to the absolute biological requirement for Thg1 activity for tRNAHis function in eukaryotes including humans, the recently demonstrated link between Thg1 overexpression and diabetic nephropathy, and the possibility of discovering novel pathways of mitochondrial 5'-tRNA editing and/or repair, defects in which could contribute to the pathology of human diseases. Moreover, a detailed understanding of the mechanism of Thg1 catalysis in diverse organisms may lead to identification of unique properties of Thg1 homologs from significant human pathogens, such as Plasmodium falciparum and Trichomonas vaginalis, which can be targeted for the development of new antiparasitic or antifungal agents.

描述（由申请人提供）： 项目摘要/摘要：tRNAHis 鸟苷基转移酶 (Thg1) 在酵母中是绝对必需的，并且可能在所有真核生物中都是必需的，因为在所有已研究的真核生物中，tRNAHis 上对 G-1 都有普遍的需求。 Thg1 通过一种不寻常的非模板化 3'-5' 核苷酸添加反应，通过一种未知的催化机制将 G-1 添加到 tRNAHis 上，而这种机制无法根据与已知酶的相似性进行预测，因此很可能采用一种新的催化机制。此外，我们最近证明所有 Thg1 家族成员都能催化与各种底物的模板依赖性 3'-5' 加成反应，并且这种活性用于古细菌中的 G-1 加成形式，以及原生动物中不寻常的 tRNA 编辑反应。这些模板化 3'-5' 添加的作用极大地扩展了 Thg1 家族成员所表现出的催化活性范围。尽管如此，在不需要酶促 G-1 添加到 tRNAHis 的古细菌和细菌中存在 Thg1 同源物，并且在酵母和人类细胞中存在无法解释的 Thg1 相关表型，这表明 3'-5' 添加的其他作用尚未被发现。该申请提出使用动力学、遗传、生化和结构技术来研究由不同Thg1家族成员催化的非模板化和模板化3'-5'加成反应的分子机制和生物学功能。这些结果将为生物学中一种新颖且明显广泛但很大程度上未经探索的反应的催化提供深入的了解，并将有助于进一步研究生物系统中 3'-5' 核苷酸添加的替代功能。 公共卫生相关性： 项目叙述 对 Thg1 家族成员催化的不寻常 3'-5' 核苷酸加成反应的研究对人类健康具有重要意义，因为包括人类在内的真核生物中 tRNAHis 功能对 Thg1 活性的绝对生物学要求，最近证明 Thg1 过度表达与糖尿病肾病之间的联系，以及发现线粒体 5'-tRNA 编辑新途径的可能性 和/或修复可能导致人类疾病病理的缺陷。此外，对不同生物体中 Thg1 催化机制的详细了解可能有助于鉴定来自重要人类病原体（例如恶性疟原虫和阴道毛滴虫）的 Thg1 同源物的独特特性，这些特性可用于开发新的抗寄生虫或抗真菌药物。