FIG. 10.

Effect of sequential deletion of 5′-terminal nucleotides on positive-strand RNA synthesis. (A) HeLa S10 translation-replication reaction mixtures that contained 2 mM guanidine HCl, helper RNA, and the indicated template RNA were incubated at 34°C for 4 h. (B) HeLa S10 translation-replication reaction mixtures that contained 2 mM guanidine HCl and helper RNA were incubated at 34°C for 1 h. The indicated template RNA was added and incubated for 1 h. PIRCs were isolated from the reactions shown in panels A and B, resuspended in reaction mixtures containing [³²P]CTP, and incubated for 1 h at 37°C. The resulting ³²P-labeled product RNA was analyzed by gel electrophoresis and quantitated as described for Fig. 8. The ratios of positive/negative-strand synthesis shown were calculated for the wild-type and mutant RNAs as explained in Materials and Methods. The predicted secondary structures for the 5′ end of the positive strand and the 3′ end of the negative strand are shown for mutant RNAs. The deleted nucleotides are underlined.