Maf1-mediated repression of RNA polymerase III transcription inhibits tRNA degradation via RTD pathway

Maf1-7A mutant suppresses thermosensitive phenotype and stabilizes tRNA^Val(AAC) in trm4Δtrm8Δ cells. trm4Δtrm8Δmaf1Δ strain, generated as described in Materials and Methods, was transformed with empty plasmid pRS415 [CEN], pAH095 encoding wild-type Maf1 [CEN MAF1], or pAH247 encoding Maf1-7A mutant [CEN MAF1-7A]. (A) Transformants were grown overnight at 30°C in SD-ura liquid media, cultures were diluted to OD₆₀₀ = 1.0 and serially 10-fold diluted, spotted on YPD plate, and incubated at 30°C or 37°C for 2 d. (B) Cells were grown in SC-Leu at 23°C, transferred to 37°C, and harvested as indicated. RNA was isolated and analyzed by Northern hybridization with tRNA^Val(AAC) probe and 5.8S rRNA probe (loading control). For quantification of tRNA^Val(AAC) levels were normalized to 5.8S rRNA. Bars represent magnitude of tRNA^Val(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 < 0.005 for the indicated probe (**) was calculated relative to control strain transformed with empty plasmid [CEN] at 120 time point.