Dihydrouridine modification of tRNA is widely observed in
prokaryotes and eukaryotes, as well as in some archaea. In
Saccharomyces cerevisiae every sequenced tRNA has at
least one such modification, and all but one have two or more.
We have used a biochemical genomics approach to identify the
gene encoding dihydrouridine synthase 1 (Dus1, ORF YML080w),
using yeast pre-tRNAPhe as a substrate. Dus1 is a
member of a widespread family of conserved proteins, three other
members of which are found in yeast: YNR015w, YLR405w, and YLR401c.
We show that one of these proteins, Dus2, encoded by ORF YNR015w,
has activity with two other substrates: yeast
pre-tRNATyr and pre-tRNALeu. Both Dus1
and Dus2 are active as a single subunit protein expressed and
purified from Escherichia coli, and the activity of
both is stimulated in the presence of flavin adenine dinucleotide.
Dus1 modifies yeast pre-tRNAPhe in vitro at U17,
one of the two positions that are known to bear this modification
in vivo. Yeast extract from a dus1-Δ strain is completely
defective in modification of yeast pre-tRNAPhe, and
RNA isolated from dus1-Δ and dus2-Δ strains
is significantly depleted in dihydrouridine content.