Fig 8. The lethality of an S. cerevisiae trm6Δ mutant is suppressed by deletion of both MET22 and TRF4, but not by either deletion alone, and results in modest tRNA_i^Met(CAU) levels.

(A) Growth test on plates of an S. cerevisiae trm6Δ [URA3 TRM6] strain with a met22Δ and/or trf4Δ mutation. Strains were grown overnight in YPD media at 30°C, diluted to OD₆₀₀~3, serially diluted 10-fold in water, and 2 μL were spotted onto YPD or 5-FOA plates as indicated, and grown for 4 days. (B) Pie streak growth test of an S. cerevisiae trm6Δ [URA3 TRM6] strain with a met22Δ and/or trf4Δ mutation. Strains were grown overnight in YPD media at 30°C, and then 2 μl of each of the cultures was streaked on 5-FOA plates for single colonies, and then incubated at 30°C for 7 days. (C) Northern analysis of tRNA_i^Met(CAU) in S. cerevisiae trm6Δ met22Δ trf4Δ mutants. Strains were grown in YPD at 30°C for 6 hours as described in Materials and Methods, and RNA was isolated and analyzed by northern blotting. (D) Quantification of tRNA_i^Met(CAU) levels in S. cerevisiae trm6Δ met22Δ trf4Δ mutants. tRNA levels were quantified as in Fig 2B.