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The Cysteine Desulfurase IscS Is Required for Synthesis of All Five Thiolated Nucleosides Present in tRNA from Salmonella enterica Serovar Typhimurium. Kristina Nilsson, 2002.Deficiency of a modified nucleoside in tRNA often mediates suppression of +1 frameshift mutations . In Salmonella enterica serovar Typhimurium strain TR970 (hisC3737), which requires histidine for growth, a potential +1 frameshifting site, CCC-CAA-UAA, exists within the frameshifting window created by insertion of a C in the hisC gene . This site may be suppressed by peptidyl-tRNAProcmo5UGG (cmo5U is uridine-5-oxyacetic acid), making a frameshift when decoding the near-cognate codon CCC, provided that a pause occurs by, e.g., a slow entry of the tRNAGlnmnm5s2UUG (mnm5s2U is 5-methylaminomethyl-2-thiouridine) to the CAA codon located in the A site . We selected mutants of strain TR970 that were able to grow without histidine, and one such mutant (iscS51) was shown to have an amino acid substitution in the L-cysteine desulfurase IscS . Moreover, the levels of all five thiolated nucleosides 2-thiocytidine, mnm5s2U, 5-carboxymethylaminomethyl-2-thiouridine, 4-thiouridine, and N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine present in the tRNA of S . enterica were reduced in the iscS51 mutant . In logarithmically growing cells of Escherichia coli, a deletion of the iscS gene resulted in nondetectable levels of all thiolated nucleosides in tRNA except N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine, which was present at only 1.6% of the wild-type level . After prolonged incubation of cells in stationary phase, a 20% level of 2-thiocytidine and a 2% level of N-6-(4-hydroxyisopentenyl)-2-methylthioadenosine was observed, whereas no 4-thiouridine, 5-carboxymethylaminomethyl-2-thiouridine, or mnm5s2U was found . We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNAGlnmnm5s2UUG due to mnm5s2U deficiency . Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant (mnmA) lacking only mnm5s2U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm5s2U and 5-carboxymethylaminomethyl-2-thiouridine and not the lack of any of the other three thiolated nucleosides that are also absent in the iscS deletion mutant .
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