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Real-time monitoring of single ZTP riboswitches reveals a complex and kinetically controlled decision landscape

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$a The 75-nt ZTP riboswitch aptamer domain (Δterm) was thermally refolded at 0 mM ZMP and then incubated with different concentrations of ZMP. <t>Gaussian</t> fitting with global constraints was used to determine the relative population of the ZMP-bound (magenta) vs. ZMP-unbound (green) aptamer. The zero- E FRET state (blue) was also fit to account for a small fraction (~5 %) of annealed RNA due to remaining splint ssDNA from sample preparation. b Example single-molecule trajectories of thermally refolded Δterm in the absence of ZMP (top) and in the presence of 1 mM ZMP (bottom). Intensities of Cy3 (green), Cy5 (red), and the resulting E FRET (blue) are shown. c The percentage of ZMP-bound aptamer plotted against ZMP concentration and fitted to the Langmuir function for thermally refolded (black) and vectorially folded (red) Δterm. The apparent K D and maximum percentage ( P max ) were determined to be 1.3 μM and 71%, respectively, for thermally refolded and 1.5 μM and 73% for vectorially folded. d E FRET histograms of Δterm after VF at 0 mM ZMP and incubation with different concentrations of ZMP. Here, a multiple turnover VF protocol was used to maximize the amount of unwound Δterm (Online Methods). Global fits were performed as in b .$

a The E FRET histograms of the WT heteroduplex before and after ATP addition at different ZMP concentrations using VF with Rep-X. <t>Gaussian</t> fitting with global constraints was used to determine the relative populations of the initial heteroduplex (blue), terminator (green), and ZMP-bound aptamer (magenta) conformations. b Bulk single-round transcription termination experiments of ZTP riboswitch are shown in the presence of 0.2 mM NTPs and varying amounts of ZMP (0–4 mM). RT and T stand for the readthrough and terminated products, respectively. Quantification of all experiments under these conditions is shown below (mean ± standard deviation (s.d.) shown as large open circles, n = 6 independent experiments shown as small circles).

a VF histograms for WT ZTP riboswitch unwound by PcrA-X in the presence of 0.1 mM ZMP are shown with <t>Gaussian</t> fits to heteroduplex (blue), terminator (green), and aptamer (magenta) populations. b The percentage of aptamer-containing conformation obtained by VF with Rep-X (black) and PcrA-X (red) are shown in the presence of 0.01 to 1 mM ZMP. The percentage of aptamer-containing conformation obtained by VF for pause mimics at positions 40 and 55 (magenta and cyan, respectively) are shown. All values are mean ± s.d. shown as large open circles with n = 2–4 independent experiments shown as small circles and offset for clarity. c Cartoon depicting the pause mimic complex at position 54, in which a cDNA is hybridized only to nt 55–94 of the WT ZTP riboswitch. Addition of ATP initiates unwinding starting from nt 55 in this complex. d E FRET histograms of the cDNA:WT heteroduplexes mimicking a pause at position 55 before (top) and after (bottom) addition of ATP in the presence of 0.1 mM ZMP, as measured by VF with Rep-X. e E FRET histograms of the pause-mimicking heteroduplexes at position 40 before (top) and after (bottom) addition of ATP in the presence of 0.1 mM ZMP, as measured by VF with Rep-X.

a Cartoon depicting the three constructs used for rate measurement experiments, either through the loss of Cy3 intensity and PIFE (top), two PIFE peaks (middle), or the loss of FRET and Cy3 intensity (bottom). The cartoon is depicted as in Fig. , with DNA strands in gray, ZTP riboswitch-derived RNA strands in black, other DNA or RNA strands in blue, Cy3 and Cy5 dyes indicated by green and red circles, and biotin labels indicated by gray circles. b Individual single-molecule trajectory in the presence of 0.1 mM ZMP for the left scheme, using loss of Cy3 intensity and PIFE as time point indicators. The drop in Cy3 intensity is arrowed, and the PIFE peak fit by a <t>Gaussian</t> curve (blue line). c Histogram of helicase rates obtained from all such experiments. The Δt between intensity drop and PIFE peak is shown ( n = 91 molecules). d Individual single-molecule trajectory (top) and histogram of all helicase rates (bottom) for double PIFE experiment with Cy3 labels at U12 and U84 used as time point indicators. For the example trajectory, the raw data (black) and Gaussian fits of PIFE peaks (blue) are shown. The mean time difference (Δt) between PIFE peaks for experiments is 2.01 ± 0.20 s (mean ± standard error of the mean (s.e.m.), n = 95 molecules). e Individual single-molecule trajectory (top) and histogram of all helicase rates (bottom) for double PIFE experiment with Cy3 labels at U32 and U84. The mean Δt between PIFE peaks is 1.64 ± 0.15 s (mean ± s.e.m., n = 112 molecules).

a Individual real-time trajectory (left), overlay of all WT ZTP riboswitch folding trajectories (center, n = 56), and histograms of all trajectories at 0, 2, and 8 s after PIFE (right) using VF with Rep-X in the presence of 1 mM ZMP. The PIFE peaks were fit to a <t>Gaussian</t> (blue) to determine their centers for synchronizing trajectories. b Individual real-time trajectory (left), overlay of all WT ZTP riboswitch trajectories (center, n = 24), and histograms of all trajectories at 0, 2, and 8 s after PIFE (right) using VF with Rep-X in the absence of ZMP. The relative unwinding position is indicated by a red line, based on the experimentally determined Rep-X unwinding rate and the location of U32 on the template. Blue circles indicate time points corresponding to the unwinding and release of helices P1 and P2, helices P1-P4, and terminator helix PT. c Real-time trajectories of terminator mutant 81-83 A in the absence of ZMP using VF with Rep-X, showing an individual trajectory (left), all trajectories overlaid (center, n = 15), and histograms of all trajectories at 0, 2, and 8 s after PIFE (right). The time duration between ticks is 5 s. Histogram probabilities are on a log scale, with ticks of 0.1, 1, and 10 percent.

Article Snippet

"For traces containing multiple PIFE peaks (Fig. ), peak centers were determined by Gaussian fitting in Origin (OriginLab, Northampton, MA), and time differences between PIFEs (Δt) were calculated by the difference between the peak centers."

Figure Legend

"Fig. 2 a The 75-nt ZTP riboswitch aptamer domain (Δterm) was thermally refolded at 0 mM ZMP and then incubated with different concentrations of ZMP. Gaussian fitting with global constraints was used to determine the relative population of the ZMP-bound (magenta) vs. ZMP-unbound (green) aptamer. The zero- E FRET state (blue) was also fit to account for a small fraction (~5 %) of annealed RNA due to remaining splint ssDNA from sample preparation. b Example single-molecule trajectories of thermally refolded Δterm in the absence of ZMP (top) and in the presence of 1 mM ZMP (bottom). Intensities of Cy3 (green), Cy5 (red), and the resulting E FRET (blue) are shown. c The percentage of ZMP-bound aptamer plotted against ZMP concentration and fitted to the Langmuir function for thermally refolded (black) and vectorially folded (red) Δterm. The apparent K D and maximum percentage ( P max ) were determined to be 1.3 μM and 71%, respectively, for thermally refolded and 1.5 μM and 73% for vectorially folded. d E FRET histograms of Δterm after VF at 0 mM ZMP and incubation with different concentrations of ZMP. Here, a multiple turnover VF protocol was used to maximize the amount of unwound Δterm (Online Methods). Global fits were performed as in b . "