(A,E,I) Subunit rotation of S6/L9-labeled Pre- and PostHC measured at saturating RF1 and RF3–GTP concentrations (1 µM each). Grey line represents FRET distribution in the absence of RF3. (B,F,J) Contour plots representing the residence time of RF1-Cy5/RF1(GAQ)-Cy5 ribosomes labeled at protein L11 by Cy3 in the presence of excess RF3 (1 µM). Time courses were synchronized to the beginning of the FRET signal. FRET values (mean ±sd) are 0.67 ± 0.02 (B), 0.50 ± 0.03 and 0.76 ± 0.02 (F), and 0.53 ± 0.04 (I). (C,G,K) Contour plots representing the residence time of RF3-Cy3 on ribosomes labeled at protein L11 by Cy3 in the presence of excess RF1 or RF1(GAQ) (1 µM). FRET values (mean ± sd) are 0.51 ± 0.03 (C), 0.51 ± 0.03 (G), and 0.51 ± 0.03 (K). (D,H,L) Comparison of the rates of RF1 and RF3 dissociation and subunit rotation. (A–D) Interactions with PreHC. (E–H) Interactions with PostHC* obtained by puromycin treatment. (I–L) Interactions with PostHC which is formed in situ using RF1. All values are mean ± sd from three independent data sets. See also Supplementary file 1.