Poster abstracts

Poster number 63 submitted by Krishna Patel

Yeast tRNAHis guanylyltransferase: Kinetic investigation of nucleotide addition by a 3'-5' polymerase

Krishna Patel (Department of Chemistry& Biochemistry), Paul Yourik (Department of Chemistry& Biochemistry), Jane E.Jackman (Department of Chemistry& Biochemistry)

Abstract:
The tRNA ^Hisguanylyltransferase (Thg1) catalyzes an essential reaction in yeast, adding a non-templated single guanosine (G_-1) to the 5’end of tRNA^His. This G_-1 addition reaction is in sharp contrast to 5’-3’ addition catalyzed by other known DNA and RNA polymerases. In addition, Thg1 also catalyzes Watson-Crick (WC) base pair–dependent 3'-5' addition. Because of these unusual properties, understanding the molecular mechanism of nucleotide addition by Thg1 is of great interest. The aim of this work is to elucidate kinetic features that distinguish non-templated vs templated nucleotide addition. To do this, we exploited a kinetic framework previously developed to characterize specific steps of the non-templated G_-1 addition reaction. Using single turnover conditions, we determined kinetic parameters for the nucleotidyl transfer step for all possible combinations of WC and non-WC base paired additions to various tRNA^His substrates. First, we determined that G_-1 addition is the most efficient of any of the four possible N_-1 addition reactions, regardless of the identity of the nucleotide at the N₇₃ position. These data suggest that Thg1 interacts specifically with the incoming GTP nucleotide, and protein residues that participate in this recognition remain to be identified. Second, we observed a preference for addition of the correct WC base pairing nucleotide that is largely driven by an increase in the maximal rate of the reaction, rather than by inherent affinity for the incoming NTP. This mirrors the kinetic trend observed for selection of WC base pairs by canonical 5’-3’ polymerases, and suggests that the well-studied kinetic mechanism for ensuring fidelity may similarly be used by Thg1. Current studies aim to measure kinetics of nucleotide addition with TLP’S (Thg1-like proteins) from bacteria, archaea and mitochondria, which participate in tRNA 5'-end repair and are catalytically distinct from their eukaryotic counterparts.

Keywords: Thg1, tRNA, Watson-Crick base pairing