Fidelity of base-pair recognition by a 3'-5' polymerase: mechanism of the Saccharomyces cerevisiae tRNAHis guanylyltransferase.

Abstract

The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3′–5′ addition of a single nontemplated guanosine (G−1) to the 5′ end of tRNAHis. In addition to this activity, S. cerevisiae Thg1 (SceThg1) also catalyzes 3′–5′ polymerization of Watson–Crick (WC) base pairs, utilizing nucleotides in the 3′-end of a tRNA as the template for addition. Subsequent investigation revealed an entire class of enzymes related to Thg1, called Thg1-like proteins (TLPs). TLPs are found in all three domains of life and preferentially catalyze 3′–5′ polymerase activity, utilizing this unusual activity to repair tRNA, among other functions. Although both Thg1 and TLPs utilize the same chemical mechanism, the molecular basis for differences between WC-dependent (catalyzed by Thg1 and TLPs) and non-WC-dependent (catalyzed exclusively by Thg1) reactions has not been fully elucidated. Here we investigate the mechanism of base-pair recognition by 3′–5′ polymerases using transient kinetic assays, and identify Thg1-specific residues that play a role in base-pair discrimination. We reveal that, regardless of the identity of the opposing nucleotide in the RNA “template,” addition of a non-WC G−1 residue is driven by a unique kinetic preference for GTP. However, a secondary preference for forming WC base pairs is evident for all possible templating residues. Similar to canonical 5′–3′ polymerases, nucleotide addition by SceThg1 is driven by the maximal rate rather than by NTP substrate affinity. Together, these data provide new insights into the mechanism of base-pair recognition by 3′–5′ polymerases.