Fidelity of base-pair recognition by a 3'-5' polymerase: Mechanism of the Saccharomyces cerevisiae tRNAHis guanylyltransferase
- ↵* Corresponding author; email: jackman.14{at}osu.edu
Abstract
The tRNAHis guanylyltransferase (Thg1) was originally discovered in Saccharomyces cerevisiae where it catalyzes 3'-5' addition of a single non-templated 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.
Keywords
- Received January 22, 2021.
- Accepted March 23, 2021.
- Published by Cold Spring Harbor Laboratory Press for the RNA Society
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