Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8565, Japan.
Graduate School of Life Science, Hokkaido University, Sapporo 060-0810, Japan.
RNA. 2021 Jun;27(6):665-675. doi: 10.1261/rna.078287.120. Epub 2021 Mar 23.
Human tRNA guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNA, producing mature tRNA In human cells, cytoplasmic and mitochondrial tRNA have adenine (A) or cytidine (C), respectively, opposite to G Little attention has been paid to the structural requirements of incoming GTP in 3'-5' nucleotidyl addition by HsThg1. In this study, we evaluated the incorporation efficiencies of various GTP analogs by HsThg1 and compared the reaction mechanism with that of Thg1 (CaThg1). HsThg1 incorporated GTP opposite A or C in the template most efficiently. In contrast to CaThg1, HsThg1 could incorporate UTP opposite A, and guanosine diphosphate (GDP) opposite C. These results suggest that HsThg1 could transfer not only GTP, but also other NTPs, by forming Watson-Crick (WC) hydrogen bonds between the incoming NTP and the template base. On the basis of the molecular mechanism, HsThg1 succeeded in labeling the 5'-end of tRNA with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNA Structural comparison of HsThg1 with other Thg1 family enzymes suggested that the structural diversity of the carboxy-terminal domain of the Thg1 enzymes might be involved in the formation of WC base-pairing between the incoming GTP and template base. These findings provide new insights into an unidentified biological function of HsThg1 and also into the applicability of HsThg1 to the 5'-terminal modification of RNAs.
人 tRNA 鸟苷酰转移酶（HsThg1）催化鸟苷三磷酸（GTP）在 tRNA 的 5'-端（-1 位）向 3'-5'的添加，产生成熟的 tRNA。在人细胞中，细胞质和线粒体 tRNA 分别在 G 的反密码子位置含有腺嘌呤（A）或胞嘧啶（C）。人们对 HsThg1 3'-5'核苷酸添加过程中进入的 GTP 的结构要求关注较少。在这项研究中，我们评估了各种 GTP 类似物被 HsThg1 掺入的效率，并将反应机制与 CaThg1（海栖热袍菌 tRNA 鸟苷酰转移酶）进行了比较。HsThg1 最有效地将 GTP 掺入到模板的 A 或 C 反密码子位置。与 CaThg1 不同，HsThg1 可以将 UTP 掺入到 A 位置，将鸟苷二磷酸（GDP）掺入到 C 位置。这些结果表明，HsThg1 不仅可以通过在进入的 NTP 和模板碱基之间形成 Watson-Crick（WC）氢键来转移 GTP，还可以转移其他 NTP。基于分子机制，HsThg1 成功地用生物素化的 GTP 标记了 tRNA 的 5'-端。还对 HsThg1 进行了结构分析，同时存在线粒体 tRNA。HsThg1 与其他 Thg1 家族酶的结构比较表明，Thg1 酶羧基末端结构域的结构多样性可能参与了进入的 GTP 和模板碱基之间 WC 碱基对的形成。这些发现为 HsThg1 的未识别生物功能提供了新的见解，也为 HsThg1 在 RNA 5'-末端修饰中的适用性提供了新的见解。
