Figure 2. Exchange of Pol III* subassembly and DnaB occur on different timescales.
(A) Diagram illustrating the sptPALM experimental design used to measure bound-times. (B) Representative example of the focus life span for the Pol III ε subunit. (C) Representative examples of the distribution of fluorescent foci life-spans (blue bars) for Pol III ε subunit and DnaB, showing fitting of a single-exponential decay model (red line), the estimated bleaching rate in the same conditions (blue line) and the corrected estimated bound-time (purple line). Note that to improve accuracy in single-molecule detection tracks shorter than four localizations were removed in the case of ε but corrected during curve fitting, hence the lower bar near 0 s time point. ε data was collected using 500 ms exposure time and 1 s intervals (N = 143), DnaB data was collected using 2 s exposure time and 10 s intervals (N = 86). The plot for DnaB shows binned data for presentation purposes. (D) Summary of estimated average bound-times. Errors in the table represent SE.
Figure 2—figure supplement 1. Characterisation of mMaple fusions.
(A) Plot showing growth curves of the AB1157 and derivative strains carrying mMaple fusions to replisome components. Experiment was done in M9-Glycerol at 37°C. Average and standard deviation of three experiments are shown. (B) Table that summarizes the results from the growth curve experiments.
Figure 2—figure supplement 2. Minimal exposure to 405 nm activation light allows continuation of cell growth.
(A) Images obtained from an sptPALM experiment of a strain carrying mMaple-DnaB using 2 s exposure times of the 561 nm laser and 2 min intervals (time in minutes). Note the growth of cells despite exposure to a single event of 405 nm wavelength activation and multiple exposures to 561 nm wavelength light. Scale bar = 2 µm. (B) Plot showing lengths of cells over time for eight different cells. The average of doubling-time is similar to the generation time of AB1157 at 22°C.
Figure 2—figure supplement 3. Estimation of photoblinking, test for two binding kinetic regimes and characterisation of the effect of longer 2 s capture rates in our estimation of bound-times.
(A) Frequency of detected short gaps, likely representing photoblinking, during the tracking of a population of LacI-mMaple molecules using 500 ms capture rates. We applied a cut-off threshold at 2.6 s for the maximum duration of photoblinking based on previous characterisation of mMaple (Durisic et al., 2014). More than 75% of the molecules did not show photoblinking (N = 148 molecules). (B) Distribution of gap times between subsequent localizations at the same location of the field of view. Note that most events lasted for only one frame. N = 60 events. (C) We fitted the distribution of gap times to a single exponential function using a truncated form of MLE. This was done to account for the fraction of events shorter than 500 ms, which would be missed in our experiments. Using a 1-frame memory parameter we estimate that our analysis will prematurely terminate less than 7.5%, 3% and 0.0001% of the tracks due to blinking when using a 1 s, 2 s, and 5 s intervals, respectively. (D) Semi-log plots of the data presented in Figure 2C for ε and DnaB. The plots show a relatively linear relation between number of cases and time, which is indicative of a single regime of binding for both subunits. Further support of a single binding behaviour is presented in Supplementary file 1C. (E) Plots showing the PDF curves of bound, bleaching, and tracking times for representative results from a single experiment of ε imaged with 500 ms (left) (N = 143) and 2 s exposure (right) (N = 415). The bound-time was 7.44 s (SE ±1.07 s) and 12.34 s (SE ±1.36 s) for 500 ms and 2 s, respectively. The plot for the 500 ms example is presented to facilitate comparison and is identical to that in Figure 2C.
Figure 2—figure supplement 4. Slow diffusion of DnaB helicase complicates correct assignment of immobile molecules at sub-second capture rates.
(A–F) Analysis from PALM experiments of ε-mMaple and mMaple-DnaB using a 21 ms capture rate. (A) Calculated MSDs for the two proteins. In both cases, curves plateau at around 600 nm in agreement with the dimension of the short axis of the cell. (B) Comparison of step lengths in the tracks of diffusing molecules analysed. (C–D) Examples of detected tracks for ε and DnaB. Lines of different colours are used to facilitate the observation of individual tracks. A red line was only used to show the position of tracks representing immobile tracks where the apparent diffusion coefficient is close to 0. The outline of the cell is shown in grey. (E–F) Distribution of the apparent diffusion coefficients calculated. 2344 tracks obtained from 77 cells and 2467 tracks obtained from 90 cells were used for ε and DnaB, respectively. (G) Distribution of mean PSFs for the x-axis for tracks of ε-mMaple or mMaple-DnaB obtained from experiments done with 500 ms capture rates. In the case of ε, a clear peak close to 100 nm shows the PSF dimensions of immobile molecules, while a second peak close to 200 nm represents diffusing molecules. In contrast, the dimensions of the PSFs from immobile and diffusive molecules is less clear for DnaB. The dashed line shows the threshold used to assign immobile and diffusing molecules in our analysis.