Fig. 2. Site-directed cross-linking studies to investigate whether RF2 can interact with the rsUGA at the selenocysteine incorporation site. (A) The experimental strategy. A designed mRNA is bound to the ribosome by tRNAVal located in the P site, allowing a thioU in the +1 position of the stop codon to form cross-links with the decoding RF in the A site. (B) The mRNA sequences used for the cross-linking studies (Figures 2 and 3). (C) Polyacrylamide gel separation of cross-linked complexes after RNase T1 digestion. The complexes were transferred to a nitrocellulose membrane and then detected by autoradiography (left panel) and immunodetection of RF2 (right panel). Lanes 1, 4 and 7, and lanes 3, 6 and 9 show cross-links formed in the presence or absence of RF2 and tRNAVal, respectively. Lanes 2, 5 and 8 show the cross-links formed in the presence of RF2 only. Cross-links between mRNA and RF2 (arrow) and S1 ribosomal protein are shown.