Abstract

The eukaryotic tRNA guanine transglycosylase (TGT) is an RNA modifying enzyme incorporating queuine, a hypermodified guanine derivative, into the tRNAs^{Asp,Asn,His,Tyr}. While both subunits of the functional heterodimer have been crystallized individually, much of our understanding of its dimer interface or recognition of a target RNA have been inferred from its more thoroughly studied bacterial homolog. However, since bacterial TGT, by incorporating queuine precursor preQ1, deviates not only in function, but, as a homodimer, also in its subunit architecture, any inferences regarding the subunit association of the eukaryotic heterodimer or the significance of its unique catalytically inactive subunit are based on unstable footing. Here, we report the crystal structure of human TGT in its heterodimeric form and in complex with a 25-mer stem loop RNA, enabling detailed analysis of its dimer interface and interaction with a minimal substrate RNA. Based on a model of bound tRNA we addressed a potential functional role of the catalytically inactive subunit QTRT2 by UV-crosslinking and mutagenesis experiments, identifying the two-stranded βEβF-sheet of the QTRT2 subunit as an additional RNA-binding motif.

摘要

真核生物 tRNA 鸟嘌呤转糖基酶 (TGT) 是一种 RNA 修饰酶，将一种超修饰的鸟嘌呤衍生物 queuine 结合到 tRNA ^{Asp、Asn、His、Tyr 中}. 虽然功能性异二聚体的两个亚基都已单独结晶，但我们对其二聚体界面或目标 RNA 识别的大部分理解都是从其更深入研究的细菌同源物中推断出来的。然而，由于细菌 TGT，通过结合队列前体 preQ1，不仅在功能上发生了变化，而且，作为同源二聚体，在其亚基结构上也发生了变化，任何关于真核异二聚体的亚基结合或其独特的催化失活亚基的意义的推论都是基于不稳定的基础。在这里，我们报告了人类 TGT 的异二聚体形式并与 25 聚体茎环 RNA 复合的晶体结构，从而能够详细分析其二聚体界面和与最小底物 RNA 的相互作用。