The Cysteine Desulfurase IscS Is Required for Synthesis of All Five Thiolated Nucleosides Present in tRNA from Salmonella enterica Serovar Typhimurium

Open Access
Publisher Website
Google Scholar
Abstract
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-tRNA Procmo5UGG (cmo 5 U 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 tRNA Glnmnm5s2UUG (mnm 5 s 2 U 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, mnm 5 s 2 U, 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 mnm 5 s 2 U was found. We attribute the frameshifting ability mediated by the iscS51 mutation to a slow decoding of CAA by the tRNA Glnmnm5s2UUG due to mnm 5 s 2 U deficiency. Since the growth rate of the iscS deletion mutant in rich medium was similar to that of a mutant ( mnmA ) lacking only mnm 5 s 2 U, we suggest that the major cause for the reduced growth rate of the iscS deletion mutant is the lack of mnm 5 s 2 U 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.