Figure 2.
met22 mutations suppress the temperature-sensitive growth defect of trm8-Δ trm4-Δ mutants and prevent degradation and loss of aminoacylation of tRNAVal(AAC). (A) Mutation of MET22 in the trm8-Δ trm4-Δ strain allows growth at 37°C. Strains were grown overnight in YPD at 28°C, adjusted to OD600 ∼ 1, serially 10-fold-diluted, spotted on YPD plates, and incubated at 18°C, 30°C, and 37°C, as indicated. (B) tRNAVal(AAC) levels and aminoacylation are stable in the trm8-Δ trm4-Δ met22-Δ strain. Strains were grown in YPD at 28°C to OD600 ∼ 2 and shifted to 37°C, and cells were harvested at the indicated times. Ten micrograms of RNA isolated under acidic conditions were analyzed by Northern blotting as described in the Materials and Methods. For each strain, one sample was deacylated prior to gel electrophoresis. Dashed and solid arrows indicate aminoacylated and deacylated tRNA species, respectively. Note that tRNAVal(AAC) from strains lacking TRM8 migrates faster than from other strains. (C) Quantification of the levels of tRNAVal(AAC). The ordinate shows the ratio of the levels of tRNAVal(AAC) at each time point relative to its level in the wild-type strain immediately before temperature shift (each value itself first normalized to 5S RNA). (D) Quantification of the percentage of aminoacylation of tRNAVal(AAC).