Figure 4.

Randomly selected second-site mutations that dampen mistranslation by tRNA^Ser_UGG. (A) Base changes are indicated by the colored circles, where blue indicates a mutation to adenine, green to guanine, red to cytosine, and orange to uracil. Bases colored in red are sites of modification in tRNA^Ser (Machnicka et al. 2013). (B) Heat map of the growth rates of strains containing mutations in tRNA^Ser_UGG that mistranslate at a level that supports viability. BY4742 containing tRNA^Ser_UGG variants were grown to saturation in media lacking uracil, diluted to an OD₆₀₀ of ∼0.1 in the same media and grown for 24 hr at 30°. OD₆₀₀ was measured every 15 min. Doubling time was calculated with the R package “growthcurver” (Sprouffske and Wagner 2016) and normalized to a strain containing a wild-type tRNA^Ser. (C) Heat map of heat shock induced by tRNA^Ser_UGG derivatives. BY4742 containing different tRNA^Ser_UGG variants and a fluorescence heat shock reporter were grown to saturation in media lacking uracil in biological triplicate. Cells were diluted 1:20 in the same media and grown for 6 hr. Cell densities were normalized and fluorescence measured.