FIG. 8.
Replication of RNA transcripts with an authentic PV 5′ end. (A) Preinitiation RNA replication complexes were isolated from translation-replication reactions programmed with RNA transcripts produced from constructs that did not (lane 1) or did (lane 2) contain the cis-active hammerhead ribozyme attached to the 5′ end of the poliovirus genome sequence. Preinitiation RNA replication complexes were incubated for 60 min at 37°C in reaction mixtures containing [α-32P]CTP; RNA was phenol extracted and analyzed on nondenaturing 1% agarose gels. (B) RNA transcripts produced from constructs containing the cis-active hammerhead ribozyme (5′Rz-PV-3Awt [lanes 1 and 2], 5′Rz-PV-3A-M79V [lane 3], and 5′Rz-PV-3A-M79T [lane 4[) or vRNA (lane 5) were used to program translation-replication reactions and form preinitiation RNA replication complexes. The complexes were isolated and incubated as above in the presence (lane 2) or absence (lanes 1, 3, 4, and 5) of 2 mM GuHCl, and labeled RNAs were analyzed in denaturing gels, as for Fig. 7. The replication activity of each substrate as the percentage of synthesis observed with wild-type 5′Rz-PV RNA transcript (lane 1) is shown under each lane and was measured using a PhosphorImager.