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: 8699201
• 负责人: Jane Elizabeth Jackman
• 金额: $ 28.8万
• 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 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8699201
• 关键词: Active Sites; Affect; Archaea; Bacteria; Base Pairing; Biochemical; Biological; Biological Assay; Biological Process; Biology; Catalysis; Cells; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Data; Enzyme Kinetics; Enzymes; Eukaryota; Exhibits; Family; Family member; Genetic; Genome; Histidine-Specific tRNA; Homologous Gene; Human; Investigation; Kinetics; Life; Molecular; Nucleic Acids; Nucleotides; Organism; Phenotype; Proteins; Protozoa; Reaction; Role; Small RNA; Techniques; Testing; Transfer RNA; Variant; Yeasts; abstracting; base; biological systems; enzyme activity; guanylyltransferase; in vivo; insight; member; novel; prevent; protein function; 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.

描述（由申请人提供）： 项目概要/摘要：tRNAHis鸟苷酰转移酶（Thg 1）在酵母中是绝对必需的，并且可能遍及所有真核生物，这是由于在已研究的所有真核生物中对tRNAHis上的G-1的普遍需要。Thg 1通过一种不寻常的非模板化3 '-5'核苷酸加成反应，通过一种未知的催化机制将G-1添加到tRNAHis上，这种催化机制无法根据与已知酶的相似性进行预测，因此可能采用一种新的催化机制。此外，我们最近已经证明，所有Thg 1家族成员催化模板依赖性的3 '-5'加成反应与各种底物，这种活性是用于古细菌中的G-1加成形式，以及用于原生动物中不寻常的tRNA编辑反应。模板化的3 '-5'加成的这些所证实的作用极大地扩展了Thgl家族成员所表现出的催化活性的范围。尽管如此，在古细菌和细菌中Thg 1同系物的存在，不需要酶促G-1添加到tRNAHis，以及酵母和人类细胞中无法解释的Thg 1相关表型，表明3 '-5'添加的其他作用尚未被发现。本申请提出使用动力学、遗传学、生物化学和结构技术来研究由不同Thg 1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的、明显广泛的、但在很大程度上未被探索的反应的催化提供洞察力，并将使人们能够进一步研究生物系统中3 '-5'核苷酸添加的替代功能。