FIGURE 3.

YchF does not influence the stability or assembly of ribosomes. The steady state amounts of ribosomes were analyzed in cell extracts of the indicated strains. Cells were grown on LB medium, harvested, and lysed by a French pressure cell. Expression of the plasmid-encoded ychF (pBadYchF) was induced with 0.002% arabinose. After determining the protein concentration in the crude cell extracts, equal amounts of protein were separated by SDS-PAGE and after western transfer, analyzed with antibodies against YchF, uL22, uS2, and LexA. (B) The crude cell extracts shown in panel (A) were subjected to sucrose-gradient density centrifugation (20–50% sucrose) and the individual ribosomal fraction was monitored at 256 nm for ribosomal RNA. Indicated are the 30 S, 50 S, 70 S ribosomal fractions. (C) As in panel (A), but cells were treated with 20 mM H₂O₂ for 30 min at 37°C before harvesting. (D) As in B, the cell extracts of H₂O₂ treated cells were subjected to sucrose-gradient centrifugation and the 30 S, 50 S, 70 S, and 90/100 S ribosomal fractions are indicated. A quantification of three independent experiments is shown in Supplementary Figure 4.