Figure 4

The effect of gene mistranslations on protein synthesis. A) Validation of expression of ribosomal protein genes by real time quantitative PCR (n = 3) confirmed the down regulation of ribosome biogenesis (*P < 0.05; **P < 0.01 for T90' vs. T0' two-tailed unpaired Student's t-test comparison). B) Mistranslations decreased protein synthesis. Control and mistranslating cells (12 OD₆₀₀ units) were pulse labeled with 62.5 μCi of [¹⁴C]-Leu for 30' at 30°C. Labeled cells were then disrupted and cleared protein extracts (30 μl) were applied onto paper filters for counting incorporated radioactivity using a scintillation counter. The values in the graph represent incorporated radioactivity in tetO-tRNA cells as percent relative to Control cells. The P-values for statistical comparisons (two-tailed unpaired Student's t-test) between tetO-tRNA and Control cells are shown - **P < 0.01. C) Polysomal profiles of yeast cells mistranslating at T0', T40' and T90', showing reduction in the number of polysomes engaged in mRNA translation. The relative area of the polysomal (P) vs. total area (P+M) of Control and tetO-tRNA cells at each time point and the ratios between total polysomes+monosome material (P+M) in tetO-tRNA cells and in Control cells before (T0') and after mistranslation induction (T40' and T90'), are shown.