Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

Abstract

Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells.

Keywords: RNA polymerase; protein-DNA interactions; single-molecule tracking; superresolution; transcription.

Fig. 1.

Single-molecule tracking allows mobility-based categorizing of individual RNAPs as DNA-bound or mobile. (A) Example trajectories of individual RNAPs with specifically bound molecules colored red and mobile molecules colored blue. (Scale bar, 1 µm.) (B) The distribution of apparent diffusion coefficients, D*, for 69,900 RNAP molecules in live cells can be fitted with two diffusing species (Inset), giving a ratio of 48% bound to 52% diffusing. Using these values allows a D* threshold to be determined to categorize bound (red) and mobile (blue) molecules. (C) Representative example cells show the spatial distribution of categorized RNAP trajectories colored according to their D* value. (D) The distribution of D* values for 39800 RNAP molecules in cells after incubation with rifampicin. (E) Example cells after rifampicin treatment show fewer bound molecules. Mobile RNAPs explore most of the cell volume due to the expansion of the nucleoid.

Fig. S1.

RNAP copy number. The mean RNAP copy number per cell is 2,710 ± 700. Copy number calculated from taking long PALM movies (typically 40,000 frames) with low photoactivation levels until all proteins have photobleached.

Fig. S2.

Distributions of apparent diffusion coefficients, D*, for RNAP and control protein DNA polymerase 1 (Pol1). (A) The distribution of RNAP D* values fits poorly to a single diffusing species. (B) Pol1 was used a control to determine the D* value of DNA-bound molecules. When DNA methylation damage is induced by MMS (see ref. 20), the fraction of bound molecules increases dramatically and is clearly resolvable from the pool of mobile molecules (20). Fitting this distribution with two diffusing species allows us to determine the D* value of specifically bound molecules, with D*_bound = 0.11 µm²/s. (C) Using this D*_bound value to constrain one species and allowing a second unconstrained D* species fits well to the RNAP distribution, giving two populations of 48% bound and 52% mobile (D*_mobile = 0.36 µm²/s). (D) Fitting to the RNAP distribution after rifampicin incubation gives a threefold reduction in the fraction of bound molecules to 16%. The D* of mobile molecules increases 75% from D*_mobile = 0.357 µm²/s (95% confidence interval: 0.338–0.378 µm²/s) to D*_mobile = 0.636 µm²/s (0.609−0.658 µm²/s). (E) Wild-type pol1 had 3.6% bound molecules. (F) After rifampicin incubation, the bound fraction of Pol1 shows negligable change (3.6–3.5%), indicating that the reduction in bound population seen in RNAP is due to blocked transcription. The D* for mobile population of Pol1 also increases after rifampicin incubation [40% increase from D*_mobile 1.05 (1.02–1.07) µm²/s to D*_mobile = 1.37 (1.40–1.35) µm²/s], indicating that the effect on the mobile population is due to the global decompaction of the nucleoid caused by rifampicin.

Fig. S3.

Timelapse images of chromosome compaction and decompaction. Time lapse images showing the nucleoid stained by HU-mCherry (yellow) relative to the cell membrane stained with FM4-64 (red). At t = 0, cells are added to an LB agarose slide containing either no antibiotics (Top), 50 µg/mL rifampicin (Center), or 100 µg/mL of chloramphenicol (Bottom). Cells were imaged every 10 min for a total of 40 min.

Fig. 2.

Comparison of categorized RNAPs with the distribution of DNA. (A) An example cell shown in a brightfield image (Left, used for cell segmentation), and in a SYTOX fluorescence image (Right, showing the nucleoid DNA distribution); x axis, short cell axis; y axis, long cell axis. (Scale bar, 1 µm.) (B) The distribution of mobile molecule trajectories (blue lines/bars) closely matched the distribution of DNA (green line) as shown in histogram projections along the x and y cell axes. (C) The distribution of bound RNAPs in the same example cell is more heterogeneous, and does not closely follow the DNA distribution. (D) Pair correlation of bound molecules (from 256 cells) shows a more clustered distribution than mobile molecules. (E) The average x axis distribution of molecules is measured from many cells by taking the relative distance from the cell midline through the center of the nucleoid, with −1 and 1 representing the cell membrane. Short cells between 1.6 µm and 2.5 µm long were chosen as they have a single nucleoid, centrally located along the y axis. (F) Plot of the x axis distribution of HU molecules from 256 cells shows the average DNA density is highest at the center and lowest at the cell periphery, with 29% of molecules found in the exterior 50% of the cell width (yellow highlighted area), compared with the expected distribution from molecules evenly occupying the full cylindrical cell volume (39% expected in cell periphery; dashed gray line). The distribution of mobile RNAP molecules (from 256 cells) matched extremely well to the distribution of HU.

Fig. 3.

DNA-free diffusion of RNAP. (A) Example minimal-DNA cell (Inset); temperature-sensitive DnaC mutant cells are grown at a nonpermissive temperature to give long cells with a single centrally located chromosome. Diffusion of RNAP in minimal-DNA cells (blue columns, 97,900 molecules) is much faster than wild-type cells (gray columns, 69,900 molecules). (B) Tracking RNAPs only in the DNA-free cell endcaps (green bars, 2,400 molecules) allows the free 3D diffusion to be determined. (C) Simulated molecular tracks undergoing Brownian diffusion within a confined cell endcap volume. Analyzing the tracks using the same protocols as the experimental data gives an estimated accurate D value that best matches the experimental data. Scanning through D values from 1 µm²/s to 5 µm²/s, the best value was D = 2.6 µm²/s (black dashed line in B). (D) Plot of the x axis distribution of RNAP molecules from 72 DNA-free cell endcaps; 40% of these RNAP molecules are found in the exterior 50% of the cell width (yellow highlighted area); 39% of molecules evenly occupying the full cylindrical cell volume are expected to be found in the periphery (dashed gray line).

Fig. S4.

Distribution of unconjugated PAmCherry. (A) The distribution of D* values for unconjugated PAmCherry imaged at 1-ms exposure times. A single species fit gives a D* value of 7.2 µm²/s. (B) The probability density distribution of unconjugated PAmCherry across the x axis for 32 cells; 38 ± 2% are located in the exterior 50% of the cell width (yellow highlighted area), compared with 39% expected from an even distribution.

Fig. 4.

Transcribing RNAPs cluster at the nucleoid periphery. (A) (i) An example cell growing in minimal media with trajectories of mobile (blue) and bound (red) RNAPs shows the difference in location of transcribing RNAPs compared with the mobile population. (ii) Probability density distribution of categorized RNAPs in the nucleoid across the x axis for 256 cells. Mobile RNAPs (blue line) follow the distribution of the nucleoid (HU distribution; gray line), with 29 ± 0.7% located in the exterior 50% of the cell width (yellow highlighted area). Transcribing molecules have a significantly broader distribution (P < 0.001), with 41 ± 0.9% of molecules locating in the periphery. (iii) The 3D SIM images of live cells in minimal media show that the densest regions of RNAP (red) are located at the edge of the nucleoid (blue). Projections onto the z–x axis highlight that dense regions apparently located in the center of the cell in X−Y projections are in fact above or below the central bulk of the DNA. (B) (i) Example cells showing clustering of bound RNAP molecules, with clustered RNAPs (>6 molecules) colored purple and nonclustered RNAPs (single or pairs of RNAPs) colored green. (ii) x-axis distribution of clustered and nonclustered bound RNAPs in 120 cells shows that nonclustered RNAPs are distributed throughout the nucleoid, whereas dense clusters form at the periphery (71 ± 9%). (iii) SIM images confirm that, although the RNAP distribution frequently overlaps with the DNA distribution, the densest RNAP regions locate at the cell edge (see also Movie S2). (C) (i) An example cell after rifampicin incubation. (ii) After rifampicin incubation, the width distributions of mobile and bound RNAPs become almost identical (P > 0.05), with 31 ± 0.8% of mobile molecules and 31 ± 1.6% of bound molecules found in the cell periphery. (iii) SIM images show DNA and RNAP distributions fill most of the cell volume homogeneously. (D) (i) An example cell after chloramphenicol incubation. (ii) The width distribution shows that 46 ± 1.7% of bound RNAP is found in the periphery compared with 33 ± 0.4% of mobile molecules. (iii) SIM image showing a cell with a compacted nucleoid with RNAPs located at the edge of the bulk of DNA.

Fig. S5.

Normalized 2D histogram plots showing the average spatial distribution of categorized RNAP molecules from many cells. (A) Cells in minimal media binned by cell length with short cells (1.6–2.5 µm long) having a single centrally located nucleoid, and longer cells (2.6–3.5 µm long) having two clearly separate nucleoids. A difference plot showing the distribution of bound RNAPs from which the distribution of mobile RNAPs has been subtracted highlights the bound RNAPs located at the periphery of the nucleoid. (B) After rifampicin incubation, the nucleoid expands to fill almost all of the cell volume, and separation between nucleoids is lost in long cells. Bound RNAPs no longer have a noticeably broader distribution, which is highlighted by the difference plot. (C) Chloramphenicol incubation causes nucleoid compaction, but the broader distribution of bound RNAP remains.

Fig. S6.

Distribution of clustered and nonclustered H-NS. Performing the same clustering and x-axis distribution analysis as performed on RNAP (Fig. 4B) with a control protein, H-NS, shows that dense H-NS clusters appear to form preferentially at the center of the cell x axis, with only 12% forming in the cell periphery. Nonclustered H-NS locate throughout the nucleoid with 30% located in the cell periphery, similar to the 29% of HU molecules found in the same region.

Fig. 5.

Organization of transcription in rich media. (A) The fractions of bound RNAPs determined per cell for different media. In rich media (531 cells), a significantly larger fraction of RNAP is specifically bound compared with minimal media (883 cells). Controls after chemical fixation (322 cells) and after rifampicin incubation (204 cells) show concomitant increases and decreases in bound fraction, respectively. (B) Localizing dense RNAP foci in live cells (Inset) allows them to be tracked in time-lapse experiments (Movie S5). MSDs of RNAP foci (red) and labeled DNA loci (blue). (C) Representative cells imaged with fast acquisition PALM, showing all localizations (Top), and clustered RNAPs, with number of molecules in clusters indicated (Bottom). (D) Histogram of the cluster sizes (in number of RNAPs) for 218 cells in rich media and 298 cells in minimal media. (E) Example fields of view showing the DAPI-stained nucleoids of live cells imaged with 3D SIM. Surface contour plots highlight the nucleoid structure. (F) The 3D surface renderings of RNAP−GFP (red) and DNA (DAPI, blue) distributions in example cells grown in minimal (Left) and rich (Right) media.

Fig. S7.

Diffusion and clustering of RNAP in cells grown in rich and minimal media. (A) Fitting two species to the distribution of RNAP D* values for cells grown in minimal media. (B) Two species fitting for cells grown in rich media shows a higher fraction of bound RNAPs (63%) compared with minimal media. (C) Pair correlation of bound RNAPs (from single-molecule tracking data) shows an increase in clustering for cells in rich media, verifying that that the observed increase in clustering is not only due to the increased fraction of transcribing RNAPs. (D) Pair correlation of all localizations from rapid, high-density PALM confirms that clustering increases with growth rate. The most dramatic increase in clustering occurs at distances of <150 nm, which indicates the characteristic size of clusters.

Fig. 6.

Mechanisms of gene spatial organization. (A) RNAPs and free ribosome subunits can explore the entire nucleoid search for specific nucleic acid sequences. RNAP starts transcribing a gene within the nucleoid, and the first ribosome binds to the emerging mRNA. As the polyribosome grows and other RNAPs start transcribing, entropic forces favor movement away from the bulk of the nucleoid. (B) In the case of rRNA transcription, there is no coupled translation, but rRNA operons are extremely highly transcribed. Multiple RNAPs on the same gene may also drive movement of the gene toward the periphery of the nucleoid.