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

The tRNA guanylyltransferase (Thg1) was originally discovered in where it catalyzes 3'-5' addition of a single nontemplated guanosine (G) to the 5' end of tRNA In addition to this activity, 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 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.