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Nucleic Acids Research Advance Access published online on January 16, 2009
Nucleic Acids Research, doi:10.1093/nar/gkn1023
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© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

RNA

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

Akiko Noma, Yuriko Sakaguchi and Tsutomu Suzuki*

Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Bldg. 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan

*To whom correspondence should be addressed. Tel: +81 3 5841 8752; Fax: +81 3 3816 0106; Email: ts{at}chembio.t.u-tokyo.ac.jp

Received November 14, 2008. Revised December 5, 2008. Accepted December 8, 2008.

The wobble modification in tRNAs, 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), is required for the proper decoding of NNR codons in eukaryotes. The 2-thio group confers conformational rigidity of mcm5s2U 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 mcm5s2U. 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.


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