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Journal of Bacteriology, February 2004, p. 758-766, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.758-766.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Formation of Thiolated Nucleosides Present in tRNA from Salmonella enterica serovar Typhimurium Occurs in Two Principally Distinct Pathways

Ramune Leipuviene, Qiang Qian,{dagger} and Glenn R. Björk*

Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden

Received 30 June 2003/ Accepted 22 October 2003

tRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s2C), 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2U), and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms2io6A). The levels of all of them are significantly reduced in cells with a mutated iscS gene, which encodes the cysteine desulfurase IscS, a member of the ISC machinery that is responsible for [Fe-S] cluster formation in proteins. A mutant (iscU52) was isolated that carried an amino acid substitution (S107T) in the IscU protein, which functions as a major scaffold in the formation of [Fe-S] clusters. In contrast to the iscS mutant, the iscU52 mutant showed reduced levels of only two of the thiolated nucleosides, ms2io6A (10-fold) and s2C (more than 2-fold). Deletions of the iscU, hscA, or fdx genes from the isc operon lead to a similar tRNA thiolation pattern to that seen for the iscU52 mutant. Unexpectedly, deletion of the iscA gene, coding for an alternative scaffold protein for the [Fe-S] clusters, showed a novel tRNA thiolation pattern, where the synthesis of only one thiolated nucleoside, ms2io6A, was decreased twofold. Based on our results, we suggest two principal distinct routes for thiolation of tRNA: (i) a direct sulfur transfer from IscS to the tRNA modifying enzymes ThiI and MnmA, which form s4U and the s2U moiety of (c)mnm5s2U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s2C and MiaB in the synthesis of ms2io6A) in the transfer of sulfur to the tRNA.

* Corresponding author. Mailing address: Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden. Phone: 46 90 7856756. Fax: 46 90 772630. E-mail: glenn.bjork{at}molbiol.umu.se.

{dagger} Present address: Telecommunication System Inc., Annapolis, MD 21401.

Journal of Bacteriology, February 2004, p. 758-766, Vol. 186, No. 3
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.3.758-766.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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