FIGURE 2.

Chlorolissoclimide blocks elongation. (A) Chlorolissoclimide does not inhibit initiation complex formation. Rabbit reticulocyte lysate were preincubated with 0.6 mM cycloheximide (CHX) ± 50 μM chlorolissoclimide for 5 min at 30°C. ³²P-radiolabeled CAT mRNA was then added to the reaction, and the incubation continued for an additional 10 min at 30°C. Translation complexes were resolved by centrifugation through 10%–30% glycerol gradients. Total counts recovered from each gradient and the percentage mRNA bound in 80S complexes were as follows: CAT mRNA/CHX, 17.3%; CAT mRNA/CHX + chlorolissoclimide, 18.4%. (B) Chlorolissoclimide can trap ribosomes on mRNA templates. Rabbit reticulocyte lysate were preincubated with 50 uM chlorolissoclimide for 5 min at 30°C. ³²P-radiolabeled CAT mRNA was then supplemented to the reaction, and the incubation continued for an additional 10 min at 30°C. Translation complexes were resolved by centrifugation through 10%–30% glycerol gradients. The total counts recovered from the gradient and the percentage mRNA bound in 80S complexes was 17.8%. (C) Effect of adding chlorolissoclimide to an actively translating extract. Translations in Krebs extracts were initiated in the presence of ³⁵S-methionine, and aliquots for TCA precipitation were removed at various time points. Five minutes after initiation of translation, compound (50 uM cycloheximide or 50 μM chlorolissoclimide) or vehicle (0.5% methanol) was added to the reaction (represented by a downward arrow), and the translation was continued for another 15 min with aliquots for TCA precipitation being removed at the indicated times. Each data point is the average of two TCA precipitations (performed in duplicate). (D) Chlorolissoclimide does not inhibit peptidyl transferase activity. Formation of ³⁵S-methionine-puromycin product by the 80S ribosome was performed over the course of 30 min and detected by thin layer chromatography in the presence of vehicle (0.5% methanol) or 50 μM chlorolissoclimide. The position of the reaction products (³⁵S-methionine-tRNA, free ³⁵S-methionine and ³⁵S-methionyl-puromycin) is indicated to the right.