Formation of thiolated nucleosides present in tRNA from Salmonella enterica serovar Typhimurium occurs in two principally distinct pathways.
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.
Leipuviene
Ramune
R
Department of Molecular Biology, Umeå University, S-90187 Umeå, Sweden.
Qian
Qiang
Q
Björk
Glenn R
GR
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins / Q000502:physiology; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism*; D009876:Operon; D012329:RNA, Bacterial / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics* / Q000254:growth & development; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013891:Thiouridine / Q000378:metabolism
D029968:Escherichia coli Proteins; D018840:HSP70 Heat-Shock Proteins; D007506:Iron-Sulfur Proteins; D009705:Nucleosides; D012329:RNA, Bacterial; D013438:Sulfhydryl Compounds; C085650:hscA protein, E coli; D013891:Thiouridine; D012343:RNA, Transfer