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. 2020 Mar 24;295(18):6053–6063. doi: 10.1074/jbc.RA119.012307

Figure 5.

Figure 5.

Effects of RRF depletion on ribosome pools and stop codon read-through. A, top panels, accumulation of unrecycled ribosomes and queuing of ribosomes in the RRF-depleted strain. Crude ribosomes were isolated from TSB cultures at A600∼ 1.4 (without the aTc inducer) grown at 37 °C and analyzed by 10–40% sucrose gradient sedimentation. No translational elongation inhibitor (e.g. chloramphenicol) was added throughout the experiments; thus, minimal polysomes were preserved in the WT strain. By contrast, unusually high levels of mixed polysomes and stalled ribosomes accumulated upon RRF depletion, which was also observed to a lesser extent in the hflX knockout mutant. Lower panels, conversion of polysomes into lower order ribosomal complexes by RNase A treatment. Excess RNase A (20 μg/ml) collapsed 100S ribosomes and the WT and ΔhflX polysomes, whereas a fraction of disome-like complexes in the RRF-depleted cells are resistant to RNase A. B, possible outcome of RRF depletion. RRF deficiency prevented the recycling of post-termination complexes, which in turn reduced the ribosome pools available for the formation of 100S ribosomes. C, analysis of RRF and His6-RRF production in the RRF-depleted strain and the hflX knockout mutant used in A. The deletion of hflX did not influence RRF expression. D, RRF deficiency reduced the translational capacity and promoted stop codon read-through. The fluorescence intensity of the GFP reporter plasmid (pDM4) was normalized according to the cell density and compared between the WT and RRF-depleted strains (Δfrr::Km,PTetfrr). RRF depletion reduced the expression of the WT GFP reporter. A stop codon was introduced at the Lys-54 position of the GFP to evaluate the degree of translational read-through. In the absence of the aTc inducer, ribosomes bypassed the Lys-54 stop codon, producing low levels of GFP. The addition of aTc to the Δfrr::Km,PTet-frr strain suppressed read-through. Error bars are S.D. from five biological replicates. p values were calculated by Student's unpaired t test (n = 5, ***, p < 0.001; ****, p < 0.0001; NS, not significant).