Fig. 5
Interaction of bL12 with RF1 and RF3 in state III. a Overview showing the relative position of RF1 (orange), RF3 (pale cyan), uL11 (light blue), 23S rRNA helix h33 (dark grey) and two copies of the bL12 CTD (purple) on the ribosome (light grey). b Comparison of state II position for RF3 (pale cyan), uL11 and h33 (light blue) with state III positions for RF3 (teal), uL11 and helix h33 (dark blue). In state III, RF1 domain I (orange) becomes ordered and density (grey mesh, filtered to 7 Å) for two copies of bL12 CTD (purple) are observed. c Comparison of state III positions from (b) with state IV positions for RF3 (pale cyan), uL11 and h33 (light blue). d Peptide hydrolysis by RF1 in the presence of wild type (wt, open circles) or bL12-depleted (∆bL12, closed circles) ribosomes. Pre-hydrolysis (PreHC) complexes (0.01 µM) were incubated with increasing concentrations of RF1 for 10 s at 37 °C. Solid lines represent the hyperbolic fit of the experimental points. Error bars represent the standard deviation of the mean for four technical replicates from two independent biological experiments. The apparent affinities of RF1 for wt and ∆bL12 PreHCs are 9 ± 1 and 210 ± 30 nM, respectively. e Time courses of RF1-GAQQsy9 (0.3 µM) binding to PreHCFlu (0.05 µM) prepared with wt (red) or ∆bL12 (black) ribosomes. Buffer controls are shown in salmon and grey, respectively. Traces shown are the average of four to five technical replicates
