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

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Abstract
TRNA from Salmonella enterica serovar Typhimurium contains five thiolated nucleosides, 2-thiocytidine (s 2 C), 4-thiouridine (s 4 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), and N -6-(4-hydroxyisopentenyl)-2-methylthioadenosine (ms 2 io 6 A). 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, ms 2 io 6 A (10-fold) and s 2 C (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, ms 2 io 6 A, 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 s 4 U and the s 2 U moiety of (c)mnm 5 s 2 U, respectively; and (ii) an involvement of [Fe-S] proteins (an unidentified enzyme in the synthesis of s 2 C and MiaB in the synthesis of ms 2 io 6 A) in the transfer of sulfur to the tRNA.