The presence of 5-methyluridine (m5U)
at position 54 is a ubiquitous feature of most bacterial
and eukaryotic elongator tRNAs. In this study, we have
identified and characterized the TRM2 gene that
encodes the tRNA(m5U54)methyltransferase,
responsible for the formation of this modified nucleoside
in Saccharomyces cerevisiae. Transfer RNA isolated
from TRM2-disrupted yeast strains does not contain
the m5U54 nucleoside. Moreover, a
glutathione S-transferase (GST) tagged recombinant, Trm2p,
expressed in Escherichia coli displayed tRNA(m5U54)methyltransferase
activity using as substrate tRNA isolated from a trm2
mutant strain, but not tRNA isolated from a TRM2
wild-type strain. In contrast to what is found for the
tRNA(m5U54)methyltransferase 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 activity
of the yNucR endo-exonuclease is dependent on the RAD52
gene, and does not respond to increased gene dosage of
the RNC1/NUD1 gene. In contrast, we find
that the expression of a trm2-LacZ fusion and
the activity of the tRNA(m5U54)methyltransferase
is not regulated by the RAD52 gene and does respond
on increased gene dosage of the TRM2 (RNC1/NUD1)
gene. Furthermore, there 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 the yNucR endo-exonuclease is encoded by a gene other
than TRM2.