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

描述(由申请人提供)： 项目摘要/摘要：tRNAHis鸟苷酸转移酶(Thg1)在酵母中是绝对必需的，可能在所有真核生物中也是如此，因为它在所有已被研究的真核生物中都普遍需要tRNAHis上的G-1。Thg1通过一种不寻常的非模板化3‘-5’核苷酸加成反应将G-1加入tRNAHis，其催化机制未知，无法根据与已知酶的相似性进行预测，因此可能采用了一种新的催化机制。此外，我们最近证明了Thg1家族的所有成员都能与各种底物催化依赖于模板的3‘-5’加成反应，并且这种活性在古生菌中被用于一种形式的G-1加成反应，在原生动物中也被用于一种不寻常的tRNA编辑反应。这些模板3‘-5’加成的作用极大地扩展了Thg1家族成员表现出的催化活性的范围。然而，在古生菌和细菌中存在不需要在tRNAHis中进行酶促G-1添加的Thg1同源物，以及在酵母和人类细胞中存在不明原因的Thg1相关表型，这表明3‘-5’添加的额外作用尚未被发现。本申请提出使用动力学、遗传学、生化和结构技术来研究Thg1家族不同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。这些结果将为深入了解生物中一种新的、明显广泛但在很大程度上未被探索的反应的催化作用，并将使进一步研究生物系统中3‘-5’核苷酸加成的替代功能。 公共卫生相关性： 项目叙事研究Thg1家族成员催化的不寻常的3‘-5’核苷酸加成反应对人类健康具有重要意义，因为包括人类在内的真核生物对tRNAHis功能的Thg1活性具有绝对的生物学要求，最近发现了Thg1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，其中的缺陷可能导致人类疾病的病理。此外，对Thg1在不同生物体中催化机制的详细了解可能会导致从恶性疟原虫和阴道毛滴虫等重要人类病原体中鉴定Thg1同源物的独特性质，这些特性可以作为开发新的抗寄生虫或抗真菌药物的靶点。

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.