Overproduction of EF-1A suppresses RTD in a trm8-Δ trm4-Δ strain. (A) Overexpression of TEF1 or TEF2 suppresses the temperature sensitivity of a trm8-Δ trm4-Δ strain. A trm8-Δ trm4-Δ strain was transformed with MORF plasmids expressing the indicated ORFs under PGAL control, transformants were grown overnight in S -Ura media containing raffinose, and serial 10-fold dilutions were plated on YP media containing raffinose and galactose and incubated for 2 d at the indicated temperatures. (B) Overproduction of EF-1A prevents degradation of tRNAVal(AAC) in trm8-Δ trm4-Δ mutants. Strains containing plasmids as indicated were grown at 28°C in S -Ura media containing raffinose and galactose to OD600 0.6, thiolutin (TL) was added, cells were shifted to 34°C for the times indicated, and RNA was extracted, resolved by 10% PAGE, transferred to a membrane, and hybridized to the indicated probes, as described in the Materials and Methods. RNA levels were quantified by normalizing to 5S RNA levels and then normalizing to the values in the wild type (BY4741) at time zero. Diamonds indicate wild type; squares, trm8-Δ trm4-Δ; open triangles, trm8-Δ trm4-Δ [TEF1]; and open circles, trm8-Δ trm4-Δ [TEF2].
