Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions.
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.
Noma
Akiko
A
Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
Sakaguchi
Yuriko
Y
Suzuki
Tsutomu
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D013437:Carbon-Sulfur Lyases / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D016681:Genome, Fungal; D023281:Genomics; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D013440:Sulfides / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013891:Thiouridine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
C105273:2-thiouridine; C011701:5-carbomethoxymethyl-2-thiouridine; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D013440:Sulfides; C406283:UBA4 protein, S cerevisiae; C406282:URM1 protein, S cerevisiae; C051552:persulfides; D013891:Thiouridine; D013455:Sulfur; D012343:RNA, Transfer; D013466:Sulfurtransferases; D013884:Thiosulfate Sulfurtransferase; C115912:NFS1 protein, S cerevisiae; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase