Figure 5.—

Translational defects of gua1–G388D mutants. (A) Total polysome profiles of isogenic strains Hm500 (gua1–G388D) and Hm512 (GUA1) grown in liquid SD to midlogarithmic phase at 28° (OD₆₀₀ ∼0.8). Cycloheximide was added at 100 μg/ml before harvesting cells, and whole-cell extracts (WCE) containing ribosomes and polyribosomes prepared in the presence of 10 mm Mg⁺² and separated by velocity sedimentation on 7–50% sucrose gradients. Peaks representing free-ribosomal 40S and 60S subunits and 80S monosomes are indicated. The polysome to monosome ratio (P/M) was estimated, and the average of values from three independent determinations indicated below the A₂₅₄ tracings. (B) Polysome profiles of the same strains as in A were obtained before and after subjecting cells to glucose starvation to inhibit translation initiation (Ashe et al. 2000). Aliquots were harvested of cells growing in liquid SD to midlogarithmic phase at 28° (0 min) and after being resuspended for 2 or 5 min in glucose-free SD medium and incubated at 28°. Cycloheximide was added at 100 μg/ml 5 min before harvesting cells and the rates of polysomal runoff estimated relative to the polysomal content of each strain before glucose starvation (0 min) that was set to 100%. The polysome to monosome ratio (P/M) was calculated and the average of values from two independent determinations indicated below the A₂₅₄ tracings. (C) The same strains were cultured as described in A, but WCE were prepared in the absence of cycloheximide and Mg⁺², and resolved by velocity sedimentation through 7–50% sucrose gradients. The mean ratios of total 60S/40S subunits determined from two replicate experiments are indicated below the A₂₅₄ tracings (left). Total RNAs obtained from Hm512 (WT) and Hm500 (mutant, m) grown at 28° to midlogarithmic phase and after being transferred to 37° for 3 hr electrophoresed in a native agarose 1.1% gel. Electrophoretograms of the same RNA samples were obtained with an Agilent Technologies 2100 Bioanalyzer and the estimated 25S/18S ratios are indicated below (right).