Overproduction of Maf1 suppresses thermosensitive phenotype and stabilizes hypomodified tRNAVal(AAC) in trm4Δtrm8Δ cells. (A) trm4Δtrm8Δ cells were transformed with multicopy pFL44L plasmid with MAF1 [2μ MAF1] or empty plasmid [2μ]. Transformants were grown overnight at 30°C in SD-ura liquid media, diluted to OD600 = 1.0 and serially 10-fold diluted, spotted on YPD plates, and incubated at 30°C or 37°C for 2 d. (B) trm4Δtrm8Δ cells harboring control empty plasmid [2μ] or plasmid with MAF1 [2μ MAF1] were grown in SC-ura medium at 30°C to OD = 1.0, shifted to 37°C, and incubated for the indicated time. RNA was isolated and analyzed by Northern hybridization with tRNAVal(AAC) probe and 5.8S rRNA probe (loading control). For quantification of tRNAVal(AAC) levels were normalized to 5.8S rRNA. Bars represent magnitude of tRNAVal(AAC) change calculated relative to expression in trm4Δtrm8Δ cells bearing empty plasmid at 0 time point. SD was calculated on the basis of three independent experiments. P-value < 0.01 for the indicated probe (*) was calculated relative to control strain transformed with empty plasmid [2μ] at 120 time point. (C) trm4Δtrm8Δ transformants with multicopy plasmid carrying gene coding tRNAVal(AAC) [2μ tRNAVal(AAC)] or empty plasmid [2μ] were grown at 23°C and shifted to 37°C for 2 h, RNA was isolated and analyzed by Northern hybridization as in B. Relative levels of tRNAVal(AAC) were quantified (note that the scale is different than in B).
