fusA mutations substantially reduce error cluster formation
Figure 5A: Misreading errors and error clusters in Apr-treated wt and P610L cells. Cells were treated at 0.25 OD600 with increasing concentrations of Apr (4-256 µM), and misreading for single errors (examined in 12 missense peptides) and error cluster formation (double errors in 14 missense peptides) were quantified at various incubation times by PRM and label free quantification. The means of their median error frequencies from 3 biological replicates are illustrated by color coding, as indicated. At high error burden all missense peptides were confidently detected (rdot products > 0.97; see Supplementary Figure S8A). To provide an orthogonal measure of misreading across all conditions, the number of detected missense peptides (rdot product > 0.8) for each conditions is represented by the size of the circles (1-12 for single errors and 1-14 for double errors). The apparently lower error frequency in wt cells at high Apr concentrations is due to massive cell death and the rapid decline in actively growing cells.
Figure 5B: Growth curves of Apr-treated wt and P610L cells used to evaluate translation errors in (A). Cell growth was recorded photometrically. Shown are mean values ± SD of 3 biological replicates.
Figure 5C: Examples of correlations between error frequencies (Ef) of the initial misreading event and of error cluster formation across 189 biological conditions (from Figure 5A). Notably, the ratio depends on the error pair, but is independent of the incubation time and Apr concentration. fusA mutations suppress error cluster formation across all conditions.
Figure S8A: PRM traces of missense peptides with first errors and corresponding error clusters at induced conditions (wt 8-16 µM; P610L 128-256µM). The conditions were chosen to ensure similar levels of single substituted peptides in wt and mutant strains. Missense peptides were identified by the high sequence coverage, the coelution and identical fragmentation as of the spiked-in isotope-labeled reference peptides (rdot products >0.97). While misssense peptides with short error clusters are often high abundant in wt an P610L cells, error clusters with more amino acid residues between the consecutive errors were strongly suppressed in P610L.
