Fig 4. Mutations in the GAAC pathway suppress the temperature sensitivity of S. pombe trm8Δ mutants and restore tP(AGG) and tY(GUA) levels.

(A) gcn1, gcn2, and tif221 mutations each restored growth of trm8Δ mutants at high temperature. Strains as indicated were grown overnight in YES media at 30°C and analyzed for growth as in Fig 1B. (B) Expression of gcn2⁺ and tif221⁺ complemented the suppression phenotype of trm8Δ gcn2-1 and trm8Δ tif221-2 mutants respectively. S. pombe trm8Δ gcn2-1 and trm8Δ tif221-2 mutants expressing gcn2⁺ and tif221⁺ respectively, or a vector, were grown overnight in EMMC-Leu media at 30°C, and analyzed for growth as in Fig 1B. Note that expression of gcn2⁺ was kept to modest levels by adding thiamine to the media to partially suppress overexpression from the P_nmt1** promoter. (C) gcn1, gcn2, and tif221 mutations each partially restored tY(GUA) and tP(AGG) levels of trm8Δ mutants. Strains were grown in YES media at 30°C and shifted to 38.5°C for 8 hours as described in Materials and Methods, and RNA was isolated and analyzed by northern blotting. (D) Quantification of tRNA levels of trm8Δ GAAC mutants shown in Fig 4C. tRNA levels were quantified as in Fig 2B. tT(AGU), brown; tP(AGA), purple; tY(GUA), gray.