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A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA

Published online by Cambridge University Press:  24 April 2002

FENG XING ,
MARK R. MARTZEN  and
ERIC M. PHIZICKY
FENG XING
Affiliation:
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA
MARK R. MARTZEN
Affiliation:
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA Present address: CEPTYR, Inc., 22215 26th Avenue S.E., Bothell, Washington 98021, USA.
ERIC M. PHIZICKY
Affiliation:
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA
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Abstract

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.

Keywords

biochemical genomicsdus1tRNA processingyeast
Type
Research Article
Information
RNA , Volume 8 , Issue 3 , March 2002 , pp. 370 - 381
Copyright
© 2002 RNA Society

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