Figure 5

Characterization of TAE2, TAE3 and TAE4 deletions. (A) Total protein synthesis was measured using [³⁵S] methionine incorporation in wild type, tae2Δ, tae3Δ and tae4Δ strains. The average count for [³⁵S] methionine incorporation for wild type was 11,356,073 (± 1,400,000) counts, which is set to 100%. On average, in the absence of Tae2p, Tae3p and Tae4p, [³⁵S] methionine incorporation was reduced by approximately 30, 14 and 10%, respectively. (B) The efficiency of protein synthesis was measured using an inducible β-galactosidase reporter construct (p416). The average β-galactosidase activity for wild type was 7.5 (± 0.6) units, which is set to 100%. The β-galactosidase activity was measured after 4 h induction. Deletion of TAE2, TAE3 and TAE4 limited the expression of β-galactosidase to 13, 21 and 17% of that in wild type, respectively. (C) Deletion of TAE2, TAE3 and TAE4 resulted in increased levels of β-galactosidase from lacZ reporters with different premature stop codons (pUKC817 and pUKC818). The activity of β-galactosidase was determined by normalizing the activity of the mutant (pUKC817 and pUKC818) to the control (pUKC815). pUKC815 is the background construct without a premature stop codon and is used as a control. Bars represent standard deviations for the means. ( D) Ribosome profile analysis of yeast deletion strains tae3Δ and tae4Δ compared to wild type. Deletion of TAE3 decreased the levels of polysomes and increased free 60 S subunits. Deletion of TAE4 caused an increase in free 60 S subunits and a slight decrease in larger polysomes. Each experiment was repeated a minimum of three times. Ratios of free 60S:40 S were calculated from the areas under the curves.