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. 2021 Oct 11;10:e70090. doi: 10.7554/eLife.70090

Figure 2. Single-molecule promoter unwinding-induced fluorescence enhancement (smUIFE) in the presence of an inhibitor that prevents RNAP clamp opening: preventing RNAP clamp opening does not prevent DNA unwinding.

(A) Design of promoter unwinding experiment in presence of myxopyronin (Myx). Blue sphere, Myx; rest as in 1 A. (B) (Left) Time trajectory of intensity from Cy3 on upstream (top) and downstream (bottom) segment of promoter bubble. Colours as in 1B. Frame rates: 50 ms. Laser powers: 0.60 mW. (Right) Dwell-time histograms of promoter state before unwinding, tUNWIND.

Figure 2—source data 1. Data for single-molecule UIFE experiments in presence of Myxopyronin in Figure 2.

Figure 2.

Figure 2—figure supplement 1. Characterisation of complexes formed between RNAP and promoter fragments in presence of myxopyronin (Myx).

Figure 2—figure supplement 1.

(A) Raw images showing field of view (24 μm × 24 μm) with immobilised biotin promoter fragment bound to labelled RNAP complexes formed in presence of Myx (left), and with remaining immobilised hexahistidine-tagged RNAP holoenzyme complexes bound to Cy3-labelled promoter fragment, formed in presence of Myx, after addition of heparin (right). (B) Mean number of localisations per field of view for single Cy3-labelled promoter fragments bound to immobilised hexahistidine-tagged RNAP holoenzyme, formed in presence of Myx, before and after addition of heparin. Mean number of localisations are average of three measurements. Error bars represent the standard deviation from the mean. (C) Hidden Markov model (HMM)-assigned histograms and Gaussian fits of fluorescence intensities corresponding to pre-unwinding (yellow) and post-unwinding (green) states for promoter fragments with Cy3 on +2 position of the non-template strand (top) or –9 position of the template strand (bottom) of the promoter bubble. Mean intensities for the two states and fold-increase of intensities are shown (inset). (D) Histogram and Gaussian fit of E* showing mean E* values for clamp conformational state for clamp-labelled RNAP molecules bound to immobilised lacCONS-promoter fragments in presence of Myx.