Figure 7.

The 5′ and 3′ cleavages in HCV RNA occur in a single structural motif. Kinetic analysis of E.coli RNAse III cleavage of HCV RNA in the presence or absence of a complementary RNA oligonucleotide. Left panel: autoradiogram of RNase III cleavage of internally labeled S₁ HCV RNA transcript in the presence (+) or in the absence (−) of a synthetic RNA oligonucleotide complementary to HCV nt 23–43 (5′-GGAGUGAUCUAUGGUGGAGUG-3′). HCV RNA substrate (0.6 nM final concentration) was pre-heated at 90°C for 1 min, before the addition of reaction buffer [10 mM HEPES–KOH, pH 7.5, 10 mM Mg(OAc)₂ and 100 mM NH₄(OAc)] and left to cool down to room temperature. Cleavage reactions were performed with 20 U RNAsin, and 0.0005 U/µl (final concentration) of E.coli RNase III, in the presence of 1 µg/µl of yeast tRNA, and carried out at 37°C in a volume of 10 µl for 1 h. These optimal conditions were used in all of the experiments. In (+) reactions the synthetic complementary RNA oligonucleotide was added to a final concentration of 15 nM. Lane 13 (‘M’) contains RNA molecular weight markers composed of transcripts with a length of 82, 99, 110, 400 and 570 (S₁) nt. Lanes starting from the left represent sequentially 10, 20, 30, 40, 50 and 60 min of incubation with RNase III without (−) or with (+) complementary RNA oligonucleotide. Cleavage products were separated on 4% denaturing polyacrylamide gels and visualized by autoradiography. The arrows on the left indicate the products of the RNase III cleavage directed by the complementary oligonucleotide and on the right; the products of RNase III alone (P1+P2, P2 and P3) are indicated. Quantitation was performed with a Radioisotopic Image Analyzer. Right panel: Graphic representation of the time course processing of S₁ by E.coli RNAse III in the absence of the complementary RNA oligonucleotide.