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.
Alamgir et al. BMC Chemical Biology 2010 10:6 doi:10.1186/1472-6769-10-6