Multicopy suppressors of trm4Δtrm8Δ stabilize hypomodified tRNAVal(AAC). trm4Δtrm8Δ cells transformed with multicopy plasmid carrying either VAS1 [2μ VAS1] or TEF1 [2μ TEF1] 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 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. Standard deviation (SD) was calculated on the basis of three independent experiments. P-values—P < 0.01 for the probe indicated with a single asterisk (*) and P < 0.005 for the probe indicated with two asterisks (**)—were calculated relative to control strain transformed with empty plasmid [2μ] at 120 time point.
