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. 2012 Oct;18(10):1886–1896. doi: 10.1261/rna.033654.112

FIGURE 3.

FIGURE 3.

Reduced levels of EF-1A exacerbate the temperature sensitivity of strains subject to RTD and result in reduced levels of tRNAs subject to RTD. (A) A tef1-Δ mutation exacerbates the temperature sensitivity of trm8-Δ trm4-Δ mutants and tan1-Δ trm44-Δ mutants, and this growth defect is fully suppressed by a met22-Δ mutation. Strains were grown overnight in YPD media, serially 10-fold diluted, plated on YPD plates, and incubated for 2 d at the indicated temperatures. (B) trm8-Δ trm4-Δ tef1-Δ mutants have reduced levels of tRNAVal(AAC) and its charged species during RTD, relative to trm8-Δ trm4-Δ mutants. Strains were grown at 28°C and shifted to 32°C, cells were harvested at the times indicated, and RNA was isolated under acidic conditions to preserve charging. Then RNAs were resolved by 6.5% acidic PAGE as described in the Materials and Methods, transferred, and hybridized to probes as indicated. RNA levels were quantified as described in Figure 2B. Quantification of charging was done for each individual tRNAVal(AAC) sample. (C) tan1-Δ trm44-Δ tef1-Δ mutants have reduced levels of tRNASer(CGA) and tRNASer(UGA) relative to tan1-Δ trm44-Δ mutants during RTD. Strains were grown at 28°C, treated with thiolutin (TL), and shifted to 37°C, and RNA was analyzed at the times indicated and quantified as described in Figure 1B. Bars in black indicate 0 min; light gray, 90 min; and dark gray, 180 min. (D) A tef1-Δ mutation exacerbates the temperature sensitivity of trm8-Δ pus7-Δ mutants and trm8-Δ dus3-Δ mutants. Strains were grown, diluted, and plated on YPD plates as described in A.