Rearrangement and packing of the switch loop in the RF2 termination complex. (A) Superposition of domain 2 of free RF2 (pink; ref. 4) on that of ribosome-bound RF2 (yellow; this work). Rearrangement of the switch loop (residues 310–324; red in free RF2 and orange in bound RF2) results in reorientation and extension of α7. (B) Packing of the switch loop in a pocket, which includes A1492 of 16S rRNA, A1913 of 23S rRNA and protein S12 directs helix α7 so that the GGQ docks in the PTC. Components are colored as in Fig. 1A.

Comparison of the structures of the RF1 and RF2 termination complexes. (A) RF2 termination complex (this work), showing RF2 (yellow), P-site tRNA (orange), E-site tRNA (red), mRNA (green), 16S rRNA (cyan), 23S and 5S rRNA (gray), 30S proteins (blue), and 50S proteins (magenta). (B) RF1 termination complex (9); molecular components are colored similarly as in A. (C) RF2 in its ribosome-bound conformation, rotated ≈180° from the view shown in A, with domains numbered. The GGQ and SPF motifs are shown in red, and the switch loop is shown in orange. (D) RF1 in its ribosome-bound conformation. The GGQ and PVT motifs are indicated in red and the switch loop is shown in orange.

Interactions with the UAA stop codon in the decoding center in the RF1 and RF2 termination complexes. (A) Stereoview of the σ_A-weighted 3F_obs − 2F_calc electron density map of the stop codon and surrounding elements of RF2 and the ribosome. Electron density is contoured at 1.0 σ for RF2, and at 1.5 σ for rRNA and mRNA, and colored yellow (RF1), green (mRNA), and blue (16S rRNA). (B) Interaction of the hydroxyl group of Ser-206 of the SPF motif of RF2 with A2 of the stop codon. (C) Interaction of Thr-216 of RF2 with A3 of the stop codon. (D) Comparison of the positions of Thr-186 of the PVT motif of RF1 (gray) and Ser-206 of the SPF motif of RF2 (yellow), showing their different modes of recognition of U1 and A2 of the UAA stop codon. The structures of the two termination complexes were globally superimposed. (E) Packing of RF2 around A3 of the UAA stop codon. Val-203 would be positioned to exclude water from H-bonding to O6 of guanine, helping to discriminate against guanine at position 3. The structure model is represented as Van der Waals surfaces for RF2 (yellow) and mRNA (green); 16S rRNA is shown in blue.

Interactions of the GGQ region of RF2 at the site of catalysis. (A) σ_A-weighted 3F_obs − 2F_calc electron density for RF2 (yellow), P-site tRNA (orange) and 23S rRNA (gray), contoured at 1.7 σ. (B) Orientation of Gln-253 of the RF2 GGQ motif. The backbone amide nitrogen of Gln-253 is positioned to H-bond with the 3′-OH of A76 of P-site tRNA, whereas its side chain is oriented away from the reaction site. (C) The Q235P mutation abolishes peptide release activity of E. coli RF1. Model 70S termination complexes assembled with [³⁵S]fMet-tRNA^fMet in the P site and mRNA M0–27 (which contains an AUG codon followed a UAA stop codon), were incubated at 37 °C with (open squares) wild-type RF1, or the RF1 mutants (diamonds) Q235A (filled squares) Q235D (triangles) Q235N (filled circles) Q235P, or (open circles) no RF1. Peptide release was monitored by measuring the amount of [³⁵S]fMet extracted into ethyl acetate at the indicated time points. Error bars indicate the range of values from 2 or 3 independent experiments, which were averaged and fit to single-exponential curves to determine the rates of peptide release. (D) Mutant Q235P RF1 binds normally in a stop-codon-dependent manner. Lanes 1 and 2, neither wild-type nor Q235P RF1 binds to ribosomes in the absence of mRNA. Lanes 3 and 4, in the presence of mRNA MO-27, both Q235P and wild-type RF1 bind stoichiometrically to ribosomes.