Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions

Nucleic Acids Res. 2009 Mar;37(4):1335-52. doi: 10.1093/nar/gkn1023. Epub 2009 Jan 16.


The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm(5)s(2)U by largely fixing the C3'-endo ribose puckering, ensuring stable and accurate codon-anticodon pairing. We have identified five genes in Saccharomyces cerevisiae, YIL008w (URM1), YHR111w (UBA4), YOR251c (TUM1), YNL119w (NCS2) and YGL211w (NCS6), that are required for 2-thiolation of mcm(5)s(2)U. An in vitro sulfur transfer experiment revealed that Tum1p stimulated the cysteine desulfurase of Nfs1p, and accepted persulfide sulfurs from Nfs1p. URM1 is a ubiquitin-related modifier, and UBA4 is an E1-like enzyme involved in protein urmylation. The carboxy-terminus of Urm1p was activated as an acyl-adenylate (-COAMP), then thiocarboxylated (-COSH) by Uba4p. The activated thiocarboxylate can be utilized in the subsequent reactions for 2-thiouridine formation, mediated by Ncs2p/Ncs6p. We could successfully reconstitute the 2-thiouridine formation in vitro using recombinant proteins. This study revealed that 2-thiouridine formation shares a pathway and chemical reactions with protein urmylation. The sulfur-flow of eukaryotic 2-thiouridine formation is distinct mechanism from the bacterial sulfur-relay system which is based on the persulfide chemistry.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Carbon-Sulfur Lyases / metabolism
  • Gene Deletion
  • Genes, Fungal
  • Genome, Fungal
  • Genomics
  • Mitochondrial Proteins
  • Molecular Sequence Data
  • Protein Structure, Tertiary
  • RNA Processing, Post-Transcriptional*
  • RNA, Transfer / chemistry*
  • RNA, Transfer / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sulfides / metabolism
  • Sulfur / chemistry
  • Sulfur / metabolism*
  • Sulfurtransferases
  • Thiosulfate Sulfurtransferase / chemistry
  • Thiosulfate Sulfurtransferase / genetics
  • Thiosulfate Sulfurtransferase / metabolism
  • Thiouridine / analogs & derivatives*
  • Thiouridine / chemistry
  • Thiouridine / metabolism


  • 2-thiouridine
  • 5-carbomethoxymethyl-2-thiouridine
  • Mitochondrial Proteins
  • Saccharomyces cerevisiae Proteins
  • Sulfides
  • UBA4 protein, S cerevisiae
  • URM1 protein, S cerevisiae
  • persulfides
  • Thiouridine
  • Sulfur
  • RNA, Transfer
  • Sulfurtransferases
  • Thiosulfate Sulfurtransferase
  • NFS1 protein, S cerevisiae
  • Carbon-Sulfur Lyases
  • cysteine desulfurase