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。

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.