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Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm5), 5-methoxycarbonylmethyl (mcm5), or 5-methoxycarbonylhydroxymethyl (mchm5) side-chain. The presence of these side-chains allows proper pairing with cognate codons, and they are particularly important in tRNA species where the U34 residue is also modified with a 2-thio (s2) group. The first step in the synthesis of the ncm5, mcm5, and mchm5 side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1, which encodes the cytosolic Cu,Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-mod-ified U34 residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNAPhe. Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells.