To analyze the presence of the Elongator-dependent tRNA anticodon modification in elp1, elp2, or elp3 mutants, two established assays were used: the zymocin sensitivity assay and the SUP4 suppression assay (62). Zymocin, a trimeric toxin secreted by so-called killer strains of Kluyveromyces lactis strains, inhibits growth of sensitive yeasts by cleaving tRNAs that contain mcm5s2U34-modified tRNA anticodons. Growth inhibition by zymocin in the growth medium or intracellular expression of the catalytic γ-toxin subunit of zymocin, a tRNA endonuclease, is abolished by mutations inactivating Elongator function. The SUP4 suppression assay makes use of the SUP4-encoded suppressor tRNA, which has an mcm5U34-modified anticodon. Strains carrying the SUP4 suppressor allele plus the ochre stop codon containing ade2-1 or can1-100 alleles provide convenient readouts for Elongator activity on medium lacking adenine or containing canavanine. The latter is a toxic arginine analog taken up by the arginine permease encoded by CAN1. Elongator deficiency results in canavanine resistance or adenine auxotrophy correlating with the degree of mcm5 modification. Zymocin or γ-toxin resistance reflects the Elongator-dependent mcm5s2 modification status. For the γ-toxin assay, the γ-toxin gene was expressed from the galactose-inducible GAL1 promoter on plasmid pLF16 (63). Transformed strains were grown overnight in single dropout medium lacking leucine, and serial dilutions of the cell suspensions were spotted on either 2% dextrose–containing (repressing conditions) or 2% galactose–containing (inducing conditions) plates and incubated for 3 days at 30°C. In the zymocin or killer eclipse assay, the zymocin-producing K. lactis strain AWJ137 (64) was inoculated next to a drop of the S. cerevisiae strains to be tested (table S2). The plates were incubated at 30°C, and the growth inhibition zone around the killer strain was analyzed in the elp1, elp2, or elp3 mutants after 2 days. Alternatively, zymocin was provided by the supernatant of strain AWJ137 grown overnight in YPD.

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