Department of Molecular Structural Biology, University of Göttingen, Göttingen, Germany.
Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
RNA Biol. 2021 Oct 15;18(sup1):382-396. doi: 10.1080/15476286.2021.1950980. Epub 2021 Jul 31.
The eukaryotic tRNA guanine transglycosylase (TGT) is an RNA modifying enzyme incorporating queuine, a hypermodified guanine derivative, into the tRNAs. 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 has been inferred from its more thoroughly studied bacterial homolog. However, since bacterial TGT, by incorporating queuine precursor preQ, 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.
真核生物 tRNA 鸟嘌呤转糖基酶（TGT）是一种 RNA 修饰酶，它将 queuine，一种高度修饰的鸟嘌呤衍生物，掺入 tRNA 中。虽然该功能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靶 RNA 识别的大部分理解都是从其研究更为透彻的细菌同源物推断出来的。然而，由于细菌 TGT 通过掺入 queuine 前体 preQ，不仅在功能上而且在其同源二聚体的亚基结构上都发生了偏离，因此关于真核异二聚体的亚基缔合或其独特的无催化活性亚基的意义的任何推断都是基于不稳定的基础。在这里，我们报告了人 TGT 异二聚体形式及其与 25 个核苷酸茎环 RNA 复合物的晶体结构，从而能够对其二聚体界面及其与最小底物 RNA 的相互作用进行详细分析。基于结合 tRNA 的模型，我们通过 UV 交联和突变实验探讨了无催化活性亚基 QTRT2 的潜在功能作用，确定了 QTRT2 亚基的两条链 βEβF-片层是另一个 RNA 结合基序。
