Rbz-containing RNAs are not cleaved when synthesized in vitro by the HCV RNA polymerase NS5B due to extensive base-pairing with the template strand. (A) Schematic depicting how NS5B and T7 polymerase transcription reactions were used to produce an N79 Rbz containing RNA. Templates were such that both sets of reactions were expected to transcribe identical RNAs, either encoding an active N79 Rbz sequence or one with a single nucleotide substitution in the active site [N79(wt) and N79(ko) respectively]. The expected sizes of RNAs produced, whether cleaved by Rbz activity or not, are shown. (B) All four [α-32P] labeled RNAs produced from the transcription reactions outlined in A were subjected to a series of treatments as detailed in the provided diagram before being run on a denaturing polyacrylamide gel. The high-salt RNaseA treatment step was used to selectively degrade single-stranded RNAs. The heat denaturation/renaturation step was used to melt dsRNA and allow single-stranded RNA folding. The asterisks indicate RNAs produced as a result of Rbz cleavage. Additional smaller bands in the NS5B transcription reactions likely arise from internal initiation. Results shown are representative of one of two experiments where initial transcription reactions were carried out at room temperature.
