FIG. 4.

Dominant effect of polypeptide chain elongation inhibitors over puromycin. Preinitiation RNA replication complexes were isolated from 50 μl of HeLa S10 translation-replication reactions that contained 2 mM guanidine and were incubated at 30°C for 5 h. The preinitiation replication complexes were resuspended in 50 μl of fresh HeLa S10 translation-replication reactions containing 10 μCi of [α-³²P]CTP as described in Fig. 2 (method 3 in Materials and Methods). The reactions also contained 2 mM guanidine HCl (lane 1); no additional drug (lane 2); 50 μg of puromycin per ml (lane 3); 10 μg of diphtheria toxin per ml plus 40 μg of NAD⁺ per ml (lane 4); 0.2 μg of ricin A chain per ml (lane 5); 200 μg of cycloheximide per ml (lane 6); 8 μg of anisomycin per ml (lane 7); 10 μg of diphtheria toxin, 40 μg of NAD⁺, and 50 μg of puromycin per ml (lane 8); 0.2 μg of ricin A chain and 50 μg of puromycin per ml (lane 9); 200 μg of cycloheximide and 50 μg of puromycin per ml (lane 10); or 8 μg of anisomycin and 50 μg of puromycin per ml (lane 11) as indicated. The reactions were incubated at 30°C for 90 min, and the radiolabeled RNA products were phenol extracted, ethanol precipitated, separated by CH₃HgOH-agarose gel electrophoresis, and visualized by autoradiography.