Figure 3. Emetine does not prevent release of puromycylated luciferase from rabbit reticulocyte ribosomes.

(A) Schematic of the real-time translation monitoring assay in rabbit reticulocyte lysate. (1) (Purple trace) Ribosomes translate the full-length luciferase mRNA and release luciferase which becomes enzymatically active and results in an increase in luminescence. (Yellow trace) Ribosomes stall at the 3’ end of a truncated luciferase mRNA and little to no luminescence is observed as the ribosome-bound luciferase peptides are in an enzymatically inactive conformation. (2) Puromycin (PM) is added to the system, stopping further translation and causing all nascent peptides to release from the ribosomes. (3) (Yellow) The luciferase rapidly folds into an enzymatically active conformation and a substantial increase in luminescence is observed. (B) Either puromycin (yellow), H₂O (purple) or a mixture of emetine (EME) and puromycin (blue) was added to a reaction containing truncated luciferase mRNA at t = 21 min. Experiment was performed in duplicate; mean traces shown as solid lines and range of replicates shaded. (C) GDPNP was added to a reaction containing truncated luciferase mRNA at t = 16 min for 5 min to inhibit translation across samples. Then, either emetine (blue, purple), anisomycin (ANS) (green) or H₂O (yellow) was added to the reaction followed by puromycin (blue, yellow, green) or H₂O (purple) 5 min later. Experiment was performed in duplicate; mean traces shown as solid lines and range of replicates shaded. Note that the experiments in (A and B), and Figure 3—figure supplement 1B were done in the same batch, and the yellow traces (PM treated) in these panels are the same.

Figure 3—figure supplement 1. Additional control experiments for lysate-based luciferase assays.

(A) Luminescence measured from translation of full-length luciferase mRNA in rabbit reticulocyte lysate. Either emetine (blue), anisomycin (green) or puromycin (yellow) was added to the reaction at indicated concentrations. Due to experimental restrictions, emetine and anisomycin were added at t = 16 min and puromycin was added at t = 22 min. Experiment performed once. (B) Luminescence measured from translation of truncated luciferase mRNA in rabbit reticulocyte lysate. Either emetine (blue), puromycin (yellow) or anisomycin (green) was added to the reaction at t = 21 min for 5 min to inhibit translation across the samples. Then, either puromycin (blue, green) or H₂O (yellow) was added to the reaction. Experiment was performed in duplicate; mean traces shown as solid lines and range of replicates shaded.