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. 2019 Sep 4;213(3):849–863. doi: 10.1534/genetics.119.302525

Figure 8.

Figure 8

tRNASer variants mistranslate at nonserine codons. (A) tti2-L187R and either a wild-type tRNASerUGA or tRNASerUCU-G9A that mistranslates serine at arginine codons were transformed into the tti2 disruption strain CY6963 with TTI2 on a URA3 plasmid. Strains were grown in media lacking histidine and leucine and plated in 10-fold serial dilutions on 5-fluoroorotic acid containing medium to select against colonies containing wild-type TTI2. (B) Wild-type tRNASerUGA, an ochre suppressor serine tRNA (tRNASerUUA) or tRNASerUUA-G9A were transformed into a tti2 disruption strain containing tti2-Q276(TAA). Strains were spotted in 10-fold serial dilutions on media lacking uracil or on YPD containing 5% ethanol. (C) Mistranslating tRNAs induce a heat-shock response. Wild-type strain (BY4742) containing a fluorescence heat shock reporter was transformed with wild-type tRNASerUGA, tRNASerUGG-G26A, tRNASerCUG-G26A, tRNASerUCU-G26A, or tRNASerGAA-G26A. Strains were grown to saturation in selective minimal medium, diluted 1:20 in the same media, and grown for 6 hr. Cell densities were normalized and fluorescence measured. Each point represents one biological replicate. (D) Strains containing either wild-type tRNASer or mistranslating serine tRNAs from C were grown to saturation in media lacking uracil, diluted to an OD600 of ∼0.1 in the same media and grown for 24 hr. OD600 was measured every 15 min. Doubling time was calculated with the R package “growthcurver” (Sprouffske and Wagner 2016). Each point represents one biological replicate.