Figure 3. Kinetics of RPo formation on the AP3 promoter.

(A) Plot showing the fluorescence signal vs. time after rapid mixing of Eco holo with Cy3-AP3 promoter (Figure 3—figure supplement 1A) in a stopped flow fluorimeter. The [RNAP] giving rise to each curve is color-coded as shown in the legend. The experimental data are shown as points. The data were fit using the three-step sequential kinetic scheme (Equation 1), yielding the parameters listed in Supplementary file 4. The curve fits are shown as solid lines. (B) Same as (A) but with Mbo holo. (C) Same as (A) but with Mbo holo+RbpA+CarD. (D) Plot showing the fluorescence signal vs. time after mixing 100 nM Mbo holo with 10 nM 2-AP-AP3 promoter (Figure 3—figure supplement 1G) in a stopped flow fluorimeter. The experimental data are shown as points. The data were fit to a single-exponential (solid black line): ${\displaystyle \mathrm{F}={\mathrm{F}}_0+({\mathrm{F}}_{\mathrm{m}\mathrm{a}\mathrm{x}}-{\mathrm{F}}_0)(1-e^{k_{app}^{exp}t})}$ yielding k_app^exp = 0.030 s⁻¹. (E) Simulation of changes in the populations of P (red), RP1 (orange), RP2 (green), and RPo (blue) under the same conditions as the experiment of panel (D). The kinetic parameters used to generate the simulation are from Supplementary file 4. The data were fit to a single-exponential (thin black line), yielding k_app^sim = 0.034 s⁻¹. (F) (top) The three-step sequential kinetic scheme that best accounts for all of the kinetic data is shown. The steps targeted by the transcription factors RbpA (orange), CarD (green), or RbpACarD together (blue) are denoted. Arrows pointing at a parameter indicate an increase in that parameter in the presence of the factor (compared to Mbo holo alone by the fold-amount shown below); the ‘T’ symbol indicates the factor reduces the parameter. The most important difference between the reference (Mbo holo alone) and Eco holo (magenta) is also illustrated. RbpA, CarD, and RbpACarD all increase k₂ significantly. CarD also reduces k_-3, as does Eco holo to a much greater extent. (bottom) Schematic free energy profile for RPo formation. The black curve represents Mbo holo alone. The colored curves illustrate the most important changes induced by the factors (RbpA, orange; CarD, green; RbpACarD, blue; Eco holo, magenta). (G) Simulations of changes in the populations of P (red), RP1 (orange), RP2 (green), and RPo (blue) when [P]₀ = 1 nM and [RNAP]₀ = 100 nM for Mbo holo alone (left) and Mbo holo+RbpA+CarD (right). The kinetic parameters used to generate the simulation are listed in Supplementary file 4. RbpA and CarD together induce a significant increase in k₂, producing a large transient burst of RP2, driving formation of RPo.

DOI: http://dx.doi.org/10.7554/eLife.22520.006

Figure 3—figure supplement 1. Cy3 promoters, association and dissociation data, activation energies, and 2-AP promoter.

(A) Schematics showing the sequences of the Cy3-AP3 and Cy3-VapB promoter constructs, illustrating the attachment points for the Cy3 label by the chemistry shown below. (B) Plot showing the fluorescence signal vs. time after rapid mixing of different concentrations of Mbo holo+RbpA with 1 nM Cy3-AP3 promoter (Figure 3—figure supplement 1A) in a stopped flow fluorimeter. The [RNAP] giving rise to each curve is color coded as shown in the legend of Figure 3A. The experimental data are shown as points. The data were fit using the three-step sequential kinetic scheme (Equation 1), yielding the parameters listed in Supplementary file 4. The curve fits are shown as solid lines. (C) Same as (B) but with Mbo holo+CarD. (D) Mbo holo RPo dissociation experiment. RPo was preformed by incubating 250 nM Mbo holo with 1 nM Cy3-AP3 DNA (Figure 3—figure supplement 1A), then mixed with 1 µM unlabeled competetive promoter trap DNA (Davis et al., 2015). The fluorescence signal was monitored as a function of time (blue data points). The thin black line shows the simulated experiment calculated fro the kinetic parameters listed in Supplementary file 4. (E) Same as (D) but with Mbo holo+RbpA. (F) The effect of RbpA and CarD on the activation energies of the kinetic steps of RPo formation on the AP3 promoter. The three-step sequential kinetic scheme is illustrated on top, with the rate constants for each step color-coded. Below, the activation energies for the different steps (color-coded) are plotted in a bar graph. (G) Sequence of the 2-AP-AP3 construct used in the experiment of Figure 3D. The introduced 2-AP is shown in magenta.

DOI: http://dx.doi.org/10.7554/eLife.22520.007