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. 2019 Apr 29;47(12):6466–6477. doi: 10.1093/nar/gkz313

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© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Nucleotide addition stabilizes the vRNA initiation state. (A) Single-molecule FRET histogram from immobilized replication complexes after ApG addition. E* represents apparent FRET efficiency, initiation state = I, pre-initiation state = P, frame time: 100 ms. (B) Example time trace showing transitions between the initiation and pre-initiation states. (C) Hidden Markov Modeling (HMM) was used to assign the states, which were colour-coded and binarized before being depicted in a single plot from shortest to longest in length. The low-FRET initiation state is represented in blue and the high-FRET pre-initiation state represented in grey. Number of traces (n) = 106. (D) Histograms of initiation (top) and pre-initiation (bottom) dwell times for immobilized replication complexes in the absence of nucleotides. The data were fitted to single (solid red line) or double-exponential (dotted red line) functions. (E) Binarized time traces from immobilized replication complexes after 500 μM ApG addition. Number of traces (n) = 102. (F) Histograms of initiation (top) and pre-initiation (bottom) dwell times for immobilized replication complexes after ApG addition.