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

RNA. 2021 Jun;27(6):683-693. doi: 10.1261/rna.078686.121. Epub 2021 Mar 31.

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.

Keywords: RNA polymerase; RNA processing; TLP; Thg1; single-turnover kinetics.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Amino Acid Sequence
  • Base Pairing
  • Crystallography, X-Ray
  • Guanosine Triphosphate / metabolism
  • Kinetics
  • Nucleotides / metabolism
  • Nucleotidyltransferases / chemistry
  • Nucleotidyltransferases / metabolism*
  • RNA, Transfer, His / metabolism
  • Saccharomyces cerevisiae / enzymology*
  • Sequence Alignment

Substances

  • Nucleotides
  • RNA, Transfer, His
  • Guanosine Triphosphate
  • Nucleotidyltransferases
  • tRNA guanylyltransferase