Abstract: Elongator complex is required for formation of the side chains at position 5 of modified nucleosides 5-carbamoylmethyluridine (ncmU), 5-methoxycarbonylmethyluridine (mcmU), and 5-methoxycarbonylmethyl-2-thiouridine (mcms(2)U) at wobble position in tRNA. These modified nucleosides are important for efficient decoding during translation. In a recent publication, Elongator complex was implicated to participate in telomeric gene silencing and DNA damage response by interacting with proliferating cell nuclear antigen (PCNA). Here we show that elevated levels of tRNA(Lys)(s(2)UUU), tRNA(Gln)(s(2)UUG), and tRNA(Glu)(s(2)UUC), which in a wild-type background contain the mcms(2)U nucleoside at position 34, suppress the defects in telomeric gene silencing and DNA damage response observed in the Elongator mutants. We also found that the reported differences in telomeric gene silencing and DNA damage response of various elp3 alleles correlated with the levels of modified nucleosides at U. Defects in telomeric gene silencing and DNA damage response are also observed in strains with the tuc2Delta mutation, which abolish the formation of the 2-thio group of the mcms(2)U nucleoside in tRNA(Lys)(mcms(2)UUU), tRNA(Gln)(mcms(2)UUG), and tRNA(Glu)(mcms(2)UUC). These observations show that Elongator complex does not directly participate in telomeric gene silencing and DNA damage response, but rather that modified nucleosides at U are important for efficient expression of gene products involved in these processes. Consistent with this notion, we found that expression of Sir4, a silent information regulator required for assembly of silent chromatin at telomeres, was decreased in the elp3Delta mutants.