Poster abstracts

Poster number 27 submitted by Jeremy Henderson

Investigation of Saccharomyces cerevisiae Trm10 tRNA methyltransferase (m1G9) activity: substrate specificity and essential amino acids for catalysis

Jeremy C. Henderson (Biochemistry, The Ohio State University), Jane E. Jackman (Biochemistry, The Ohio State University)

Abstract:
Trm10 catalyzes SAM dependent N-1 methylation of G₉ (m¹G₉) for at least 10 tRNA species in S. cerevisiae, including tRNA^Gly. The mechanism by which Trm10 methylates tRNA is unknown, and since there is no identifiable sequence homology between Trm10 and any other enzyme family, including other methyltransferases, the catalytic mechanism is likely novel. Several puzzling observations suggest that the Trm10 biological function is more complex than currently understood. Although m¹G₉ modification of tRNA is non-essential under standard biological conditions, recent results show that trm10Ä yeast strains are highly sensitive to 5-fluorouracil, a known anti-tumor agent for treatment of solid cancers; the reason for this observation is unknown. Moreover in yeast, TRM10 is a single copy gene, while multiple homologs of TRM10 are found in several higher eukaryotes, including at least three in humans.

Trm10 only modifies a subset of tRNA species in vivo. The sequence elements that define what tRNAs are substrates for Trm10 are unknown, and are not obvious from simple sequence comparison. We note, there are at least 12 other yeast tRNA species, including tRNA^Val and tRNA^Leu, that have an unmodified G₉. To investigate Trm10 substrate specificity, we have developed sensitive assays using three site-specifically labeled tRNA substrates. All of these substrates have a G residue at position 9, yet tRNA^Val and tRNA^Leu are not substrates in vivo, while tRNA^Gly is a substrate for m¹G₉ modification. Trm10 activity with these substrates is being measured under steady-state conditions to determine parameters including k_cat/K_M, an indicator of substrate specificity. These results will be used as the foundation for identification of nucleotides necessary for tRNA specificity. We are also currently investigating the roles of several highly conserved amino acids in the Trm10 reaction by mutagenesis. Analysis of the resultant effects should provide insight into the molecular basis for Trm10 activity, a potentially novel methyltransferase enzymatic mechanism.

Keywords: tRNA, Trm10, yeast