FIG. 4.

Competition assays between puromycin and Trp using different isolated TnaC-tRNA-ribosome complexes. In vitro translation reactions were performed with [³⁵S]methionine to label the TnaC peptides. The cell extracts employed were pretreated with an anti-RF2 antiserum, and biotinylated tnaC mRNAs were used in each reaction mixture. The TnaC-tRNA-ribosome-mRNA complexes were isolated from the in vitro translation reactions by using streptavidin beads, and the isolated complexes were initially incubated with (+) or without (−) 2 mM Trp at 37°C for 5 min. Later, the complexes were incubated with different concentrations of puromycin (Puro) at the same temperature for 10 additional minutes. The final products of the reaction were resolved, and their presence was determined by measuring radioactivity. (A and E) Complexes containing wild-type 23S rRNA. (B and F) Complexes containing +A751 23S rRNA. (C and G) Complexes containing U2584C 23S rRNA. (D and H) Complexes containing G2583A 23S rRNA. (I) Plot of the data in panels A to D. (J) Plot of the data in panels E to H. The percentage of the TnaC as TnaC-tRNA was calculated by dividing the cpm in each TnaC-tRNA band by the combined cpm in the TnaC-tRNA and TnaC bands.