Mechanistic characterization of the sulfur-relay system for eukaryotic 2-thiouridine biogenesis at tRNA wobble positions
Open Access
- 16 January 2009
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 37 (4), 1335-1352
- https://doi.org/10.1093/nar/gkn1023
Abstract
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.Keywords
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