Mutational perturbations of eIF4F subunits similarly reduce translation of GFP NUS1 mRNAs with short and long unstructured 5′ UTRs. GFP fluorescence measured in mutant strains normalized to respective GFP fluorescence measured in the wild-type strain. Error bars indicate standard deviations determined from three biological replicates. (A) eIF4A-encoding tif1, tif2 or eIF4B-encoding tif3 were deleted in BY4741 background as indicated. (B) Either wild-type (TIF1) or temperature-sensitive (tif1-A79V) alleles of the eIF4A-encoding tif1 were expressed in the strain lacking both chromosomal copies of tif1 and tif2. (C) An amino acid substitution L614F, which reduces eIF4G binding to eIF4A, was introduced into tif4631 in the background of tif4632 deletion to create the temperature-sensitive tif4632Δtif4631-L614F strain. (D) An amino acid substitution M570K, which reduces eIF4G binding to eIF4A, was introduced into tif4632 in the background of a tif4631 deletion to create temperature-sensitive tif4631Δtif4632-M570K strain. BY4741 strain is considered as wild type in A, C, and D. In experiments with temperature-sensitive strains tif2Δtif1-A79V and tif4632Δtif4631-L614F, the Gal 1,10 promoter was induced concurrently with raising the incubation temperature from 30°C to 37°C. Respective wild-type control strains received identical treatment.
