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. 2020 Oct 9;295(50):17200–17214. doi: 10.1074/jbc.RA120.015025

Figure 2.

Figure 2.

rRNA hydrolysis in vitro occurs independently of ribosome-associated proteins. A, schematic representation of the workflow to test the effect of removing the protein from ribosomes on rRNA cleavage in the presence of Fe2+ and ascorbic acid. Ribosomes were purified from WT cells by centrifugation through a 50% sucrose cushion, and ribosomal pellets containing 10 µg of RNA were treated with the indicated amounts of proteinase K for 5 min on ice or remained untreated. B, samples of purified ribosomes treated with proteinase K at the indicated concentrations were analyzed by SDS-PAGE and stained for total protein content with Coomassie Blue. C, samples of purified ribosomes treated with proteinase K at the indicated concentrations were treated with ascorbic acid and Fe(NH4)2(SO4)2, as in Fig. 1, and analyzed by Northern hybridization using the 25S rRNA probe y540. A representative hybridization is shown.