A conserved family of Saccharomyces cerevisiae synthases effects dihydrouridine modification of tRNA.
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-tRNA(Phe) 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-tRNA(Tyr) and pre-tRNA(Leu). 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-tRNA(Phe) in vitro at U17, one of the two positions that are known to bear this modification in vivo. Yeast extract from a dus1-A strain is completely defective in modification of yeast pre-tRNAPhe, and RNA isolated from dus1-delta and dus2-delta strains is significantly depleted in dihydrouridine content.
Xing
Feng
F
Department of Biochemistry and Biophysics, University of Rochester School of Medicine, New York 14642, USA.
Martzen
Mark R
MR
Phizicky
Eric M
EM
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016366:Open Reading Frames / Q000502:physiology; D010957:Plasmids; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics*; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics; D012356:RNA, Transfer, Leu / Q000737:chemistry / Q000378:metabolism; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000378:metabolism; D012365:RNA, Transfer, Tyr / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D005656:Fungal Proteins; D012331:RNA, Fungal; D012356:RNA, Transfer, Leu; D012360:RNA, Transfer, Phe; D012365:RNA, Transfer, Tyr; D011993:Recombinant Fusion Proteins; C007419:dihydrouracil; D006639:Histidine; D014498:Uracil; D012343:RNA, Transfer; D005982:Glutathione Transferase