Identification of the TRM2 gene encoding the tRNA(m(5)U(54)) methyltransferase of Saccharomyces cerevisiae

The presence of 5-methyluridine (m(5)U) at position 54 is a ubiquitous feat ure of most bacterial and eukaryotic elongator tRNAs. In this study, we hav e identified and characterized the TRM2 gene that encodes the tRNA(m5U54)me thyltransferase, responsible for the formation of this modified nucleoside in Saccharomyces cerevisiae. Transfer RNA isolated from TRM2-disrupted yeas t strains does not contain the m5U,, nucleoside. Moreover, a glutathione S- transferase (GST) tagged recombinant, Trm2p, expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase activity using as substrate tRNA iso lated from a trm2 mutant strain, but not tRNA isolated from a TRM2 wild-typ e strain. In contrast to what is found for the tRNA(m5U54) methyltransferas e encoding gene trmA(+) in E. coli, the TRM2 gene is not essential for cell Viability and a deletion strain shows no obvious phenotype. Surprisingly, we found that the TRM2 gene was previously identified as the RNC1/NUD1 gene , believed to encode the yNucR endo-exonuclease. The expression and activit y of the yNucR endo-exonuclease is dependent on the RAD52 gene, and does no t respond to increased gene dosage of the RNC1/NUD1 gene. In contrast, we f ind that the expression of a trm2-lacZ fusion and the activity of the tRNA( m(5)U(54))methyltransferase is not regulated by the RAD52 gene and does res pond on increased gene dosage of the TRM2 (RNC1/NUD1) gene. Furthermore, th ere was no nuclease activity associated with a GST-Trm2 recombinant protein . The purified yNucR endo-exonuclease has been reported to have an NH2-D-E- K-N-L motif, which is not found in the Trm2p. Therefore, we suggest that th e yNucR endo-exonuclease is encoded by a gene other than TRM2.