Figure 5.

The duplex region of the vRNA promoter is destabilised during initial replication. Annealed RNAs, corresponding to the 5′ (5′-AGUAGAAACAAGGAGUUU) and 3′ (3′-UCGUUUUCGUCCUCAAA) ends of the vRNA promoter, were labelled with donor and acceptor fluorophores at positions 17 on the 3′ end and 3 on the 5′ end (A), or positions 17 on the 3′ end and 6 on the 5′ end (B), and were either analysed alone (top panels), or incubated with a final concentration of 100 nM RNAP (middle panels), or 100 nM RNAP and 500 μM ApG (lower panels) before single-molecule FRET spectroscopy on diffusing molecules was carried out. Histograms from three independent experiments were merged. Ratio E* represents the uncorrected FRET efficiency, n represents the number of molecules and curves were fitted with Gaussian functions to determine the centre of the distributions.