Analysis of GTP addition in the reverse (3'-5') direction by human tRNAHis guanylyltransferase

Abstract

Human tRNA^His guanylyltransferase (HsThg1) catalyzes the 3'-5' addition of guanosine triphosphate (GTP) to the 5'-end (-1 position) of tRNA^His, producing mature tRNA^His In human cells, cytoplasmic and mitochondrial tRNA^His have adenine (A) or cytidine (C), respectively, opposite to G_-1 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 Candida albicans 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^His with biotinylated GTP. Structural analysis of HsThg1 was also performed in the presence of the mitochondrial tRNA^His 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.

Keywords: 3′-5′ polymerase; RNA modification; RNA structure; aminoacyl tRNA synthetase; transfer RNA (tRNA).

FIGURE 1.

Nucleotide addition reaction by HsThg1 onto a two-piece tRNA^His. (A) Reaction scheme of a nucleotide addition reaction with natural NTPs and various GTP analogs (XTP) and a 5′-triphosphorylated RNA fragment (5′RF) with a 3′-side RNA fragment (3′RF) containing adenine or cytosine opposite the −1 position (Y = A or C). (B,C) Primer/template assay for simultaneously measuring the kinetics of nucleotide addition reactions by HsThg1 with two types of two-piece tRNA (Y = A or C). The reactions shown are time courses of activity with 10 µM HsThg1 and 0.1 mM GTP in excess over two-piece tRNA; aliquots from each time point were analyzed on urea-PAGE gels. RNA fragments were detected by SybrGold staining. Asterisk indicates upper bands which likely correspond to the G₋₂ addition to 5′RF/3′RF-A. (D) Time course experiments of the nucleotide addition reaction of HsThg1 with 3′RF-A (blue) or 3′RF-C (red). The bars in the graphs are SD of more than two independent experiments.

FIGURE 2.

Addition activities of wild-type NTP and GTP analogs by HsThg1. (A) Natural NTPs and GTP analogs used in the nucleotide addition reaction. (B,C) The relative k_obs values of nucleotide addition reactions with various GTP analogs (0.1 mM) for the template Y = A (B), Y = C (C). Values are relative to the k_obs value of wild-type activity (GTP addition in Y = A). k_obs values of HsThg1 (this study) and CaThg1 (Nakamura et al. 2018b) are colored black and gray, respectively. The bars in the graphs are SD of more than two independent experiments.

FIGURE 3.

5′-end RNA labeling with various GTP analogs by HsThg1. (A) Reaction scheme of single or multiple 5′-end RNA labeling by HsThg1 with 3′RF-A or 3′RF-C. HsThg1 adds single or multiple modified GTP analogs (XTP) to 5′-triphosphorylated 5′RF. (B) GTP analogs used in the 5′-end labeling reaction. Positions at which the GTP can be modified are indicated with Z (position 7) or M (2′OH). (C,D) Nucleotide addition by HsThg1 with various GTP analogs onto two-piece tRNA containing 3′RF-A (C) or 3′RF-C (D). The reaction mixture was incubated with HsThg1 and 1 mM modified GTP derivatives for 60 min and analyzed using urea-PAGE gels.

FIGURE 4.

Structural comparison of Thg1 and TLP enzymes. (A) Human mitochondrial tRNA^His (hmtRNA^His) is drawn as a cloverleaf structure. hmtRNA^His contains a His-specific anticodon GUG and cytosine opposite the −1 position (Y = C). (B) Structural comparison of Thg1 enzymes: HsThg1 in the presence of hmtRNA^His (HsThg1-tetramer, this study), HsThg1-dimer (PDB ID: 3OTC) and CaThg1-free (PDB ID: 3WBZ), without tRNA^His binding. The flexibility of the β-hairpin structure at the carboxy-terminal of Thg1 is shown in red. (C) Structural comparison of Thg1 enzymes with tRNA binding: HsThg1 in the presence of hmtRNA^His (HsThg1-tetramer, this study), CaThg1-tRNA^His (PDB ID: 3WC2), and MaTLP-tRNA (PDB ID: 5AXN). The unknown electron density map (2Fo-Fc map [1.5 σ]; blue, and Fo-Fc map [2.5 σ]; green) close to the flexible β-hairpin is indicated as mesh. (D) Proposed incoming nucleotide recognition mechanism of HsThg1 organized by the interaction between the β-hairpin and the elbow region of the substrate tRNA.