FIG. 7.

Effect of puromycin and cycloheximide on negative-strand RNA synthesis within preinitiation RNA replication complexes. Preinitiation replication complexes were isolated from 50 μl of HeLa S10 translation-replication reactions containing T7-PV1(A)₈₀ RNA (rather than virion RNA) and 2 mM guanidine HCl after incubation at 30°C for 5 h (method 6 in Materials and Methods). The preinitiation complexes were resuspended in 50-μl reaction mixtures containing HeLa S10 extract, translation initiation factors, 50 μCi of [α-³²P]CTP, and 5 μM CTP_final. The individual reactions contained 2 mM guanidine (lane 1), no additional drugs (lane 2), 200 μg of cycloheximide per ml (lane 3), or 100 μg of puromycin per ml (lane 4). The reactions were incubated at 30°C for 60 min (A) or for 90 min (B), and the labeled RNA was phenol extracted and ethanol precipitated. The labeled RNA products were fractionated by CH₃HgOH-agarose gel electrophoresis. The dried gels were subjected to autoradiography for visualization of the labeled RNAs. The amount of labeled negative-strand RNA was quantitated by using a Molecular Dynamics PhosphorImager.