Template-dependent 3'-5' nucleotide addition is a shared feature of tRNAHis guanylyltransferase enzymes from multiple domains of life

Abstract

The presence of an additional 5' guanosine residue (G(-1)) is a unique feature of tRNA(His). G(-1) is incorporated posttranscriptionally in eukarya via an unusual 3'-5' nucleotide addition reaction catalyzed by the tRNA(His) guanylyltransferase (Thg1). Yeast Thg1 catalyzes an unexpected second activity: Watson-Crick-dependent 3'-5' nucleotide addition that occurs in the opposite direction to nucleotide addition by all known DNA and RNA polymerases. This discovery led to the hypothesis that there are alternative roles for Thg1 family members that take advantage of this unusual enzymatic activity. Here we show that archaeal homologs of Thg1 catalyze G(-1) addition, in vitro and in vivo in yeast, but only in a templated reaction, i.e. with tRNA(His) substrates that contain a C(73) discriminator nucleotide. Because tRNA(His) from archaea contains C(73), these findings are consistent with a physiological function for templated nucleotide addition in archaeal tRNA(His) maturation. Moreover, unlike yeast Thg1, archaeal Thg1 enzymes also exhibit a preference for template-dependent U(-1) addition to A(73)-containing tRNA(His). Taken together, these results demonstrate that Watson-Crick template-dependent 3'-5' nucleotide addition is a shared catalytic activity exhibited by Thg1 family members from multiple domains of life, and therefore, that this unusual reaction may constitute an ancestral activity present in the earliest members of the Thg1 enzyme family.

Fig. 1.

Domain-dependent incorporation of G_-1 into tRNA^His. (A) In bacteria, G_-1 is genomically encoded and retained by RNase P during 5^′-processing. (B) Thg1 adds G_-1 to eukaryal tRNA^His, and catalyzes template-dependent 3^′–5^′ addition to tRNA^His variant substrates. Nucleotides added posttranscriptionally by Thg1 are indicated by parentheses. For clarity, only the altered 3^′ end sequences of tRNA^His variant substrates are shown.

Fig. 2.

Phylogeny of archaeal Thg1 homologs. An unrooted phylogenetic tree of the archaeal Thg1 homologs was constructed with Geneious Pro software (v.4.7.5) using the neighbor-joining method with a Jukes–Cantor genetic distance model and no outgroup. The tree was built upon a ClustalW alignment (36) of Thg1 sequences from 20 of 21 Thg1-containing archaea (P. aerophilum Thg1 is similar to the other three Pyrobaculum homologs and was omitted for clarity). Organism names are colored according to the presence of G_-1 on the annotated tRNA^His; blue, G_-1 is genomically encoded; red, G_-1 is not present in the genomic sequence. The four archaea investigated in this work are indicated by boxes. The dashed line indicates the separation between crenarchaeal (Upper Left) and euryarchaeal (Lower Right) clades.

Fig. 3.

Archaeal Thg1 homologs do not efficiently add G_-1 to wild-type yeast tRNA^His. (A) Assays using 5^′-³²P-labeled tRNA^His and 1 µL each Thg1, as indicated, were performed with 0.1 mM ATP and 1.0 mM GTP as described in Methods. Lane-, buffer only. Reaction products are labeled to the right of each panel, and indicated by arrows. (B) 5^′-³²P-tRNA^Hisassay performed with 1 µL yeast or MaThg1. The presence or absence of ATP or GTP (1 mM) is denoted by (+) and (-). (C) Assays of 5^′-³²P-tRNA^His with titrations (5-fold serial dilutions, starting with approximately ~50 M enzyme) of purified yeast or MaThg1; Lane-, buffer only control reaction. (D) Scheme of steps for G_-1 addition to tRNA^His showing first, adenylylation of p-tRNA^His, followed by nucleotidyltransfer to yield G_-1-containing tRNA. The apparent block in the 2nd step of the reaction observed with archaeal Thg1 is as indicated.

Fig. 4.

Archaeal Thg1 homologs efficiently catalyze G-addition opposite C₇₃ with a tRNA^His variant substrate. Assay of purified yeast and archaeal Thg1 homologs (1 µL each) with 5^′-³²P labeled C₇₃-tRNA^His variant. Lane-, buffer only. Positions of G_-1, G_-2, and G_-3 reaction products are indicated to the right of the figure.

Fig. 5.

Archaeal Thg1 homologs support growth of the yeast thg1Δ strain in the presence of C₇₃-tRNA^His. Replica plating results with yeast thg1Δ strains transformed with LEU2 plasmids containing various Thg1 homologs (indicated to the right of the figure) and HIS3 plasmids containing various tRNA^His genes (indicated on top of each panel). Growth media are indicated below each panel; images were taken after 3–4 days of growth at 30 °C.

Fig. 6.

Archaeal Thg1 catalyzes template-dependent U_-1 addition activity with wild-type tRNA^His. (A) U_-1 addition assay with 5^′-³²P labeled tRNA^His and titrations (5-fold serial dilutions) of yeast or MaThg1. Lane-, buffer only. Identity of observed reaction products indicated to the left of the panel. (B) Initial rates of N_-1 addition to wild-type tRNA^His (2 µM) with yeast Thg1 (0.2 µM) (solid bars) or MaThg1 (0.15 µM) (hatched bars), and 1 mM of each NTP, as indicated. Note separate scale on Y-axis for each enzyme's activity.