Saccharomyces cerevisiae Thg1 Uses 5′-Pyrophosphate Removal To Control Addition of Nucleotides to tRNA^His

In eukaryotes, the tRNA^His guanylyltransferase (Thg1) catalyzes 3′–5′ addition of a single guanosine residue to the −1 position (G_–1) of tRNA^His, across from a highly conserved adenosine at position 73 (A₇₃). After addition of G_–1, Thg1 removes pyrophosphate from the tRNA 5′-end, generating 5′-monophosphorylated G_–1-containing tRNA. The presence of the 5′-monophosphorylated G_–1 residue is important for recognition of tRNA^His by its cognate histidyl-tRNA synthetase. In addition to the single-G_–1 addition reaction, Thg1 polymerizes multiple G residues to the 5′-end of tRNA^His variants. For 3′–5′ polymerization, Thg1 uses the 3′-end of the tRNA^His acceptor stem as a template. The mechanism of reverse polymerization is presumed to involve nucleophilic attack of the 3′-OH from each incoming NTP on the intact 5′-triphosphate created by the preceding nucleotide addition. The potential exists for competition between 5′-pyrophosphate removal and 3′–5′ polymerase reactions that could define the outcome of Thg1-catalyzed addition, yet the interplay between these competing reactions has not been investigated for any Thg1 enzyme. Here we establish transient kinetic assays to characterize the pyrophosphate removal versus nucleotide addition activities of yeast Thg1 with a set of tRNA^His substrates in which the identity of the N_–1:N₇₃ base pair was varied to mimic various products of the N_–1 addition reaction catalyzed by Thg1. We demonstrate that retention of the 5′-triphosphate is correlated with efficient 3′–5′ reverse polymerization. A kinetic partitioning mechanism that acts to prevent addition of nucleotides beyond the −1 position with wild-type tRNA^His is proposed.