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: 8516527
• 负责人: Jane Elizabeth Jackman
• 金额: $ 27.82万
• 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/8516527
• 关键词: Active Sites; Affect; Antifungal Agents; Antiparasitic Agents; Archaea; Bacteria; Base Pairing; Biochemical; Biological; Biological Assay; Biological Process; Biology; Catalysis; Cells; 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.

描述（由申请人提供）： 项目摘要/摘要：Trnahis guanyllyllansferase（THG1）在酵母中绝对必要，并且可能在所有真核生物中，由于对trnahis的普遍要求，在所有已研究的真核生物中对Trnahis的普遍要求。 THG1通过不知名的核苷酸添加反应，通过与已知酶相似的未知催化机制来增加Trnahis的G-1，因此可能采用一种新型的催化机制来预测。此外，我们最近证明，所有THG1家族成员都与模板依赖性的3'-5'添加反应催化与各种底物的添加反应，并且该活性用于古细菌中的G-1添加形式，以及在原生态中的不寻常的tRNA编辑反应。这些表现出了模板3'-5'的作用，大大扩大了THG1家族成员所表现出的催化活动范围。尽管如此，不需要在trnahis中添加酶促G-1的古细菌和细菌中Thg1同源物的存在，并且在酵母中无法解释的与THG1相关的表型表明，尚未发现3'-5'添加的其他作用。该应用提出了使用动力学，遗传，生化和结构技术来研究非策略和模板3'-5'的添加反应的分子机制和生物学功能，这些反应由多样化的THG1家族成员催化。这些结果将为生物学中的新型且显然没有探索的新颖而广泛但尚未探索的催化提供洞察力，并能够进一步研究生物系统中3'-5'核苷酸添加的替代功能。