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

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。 公共卫生相关性： Thg 1家族成员催化的不寻常的3 '-5'核苷酸加成反应的研究对人类健康具有重要意义，这是因为在包括人类在内的真核生物中，Thg 1活性对于tRNAHis功能的绝对生物学要求，最近证实了Thg 1过表达与糖尿病肾病之间的联系，以及发现线粒体5 '-tRNA编辑和/或修复的新途径的可能性，这些缺陷可能导致人类疾病的病理学。此外，详细了解Thg 1在不同生物体中的催化机制，可能会导致从重要的人类病原体（如恶性疟原虫和阴道毛滴虫）中识别Thg 1同源物的独特特性，这些同源物可用于开发新的抗寄生虫或抗真菌药物。

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.