Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription

Abstract

In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles.

Keywords: DNA condensation; Dps; RNA polymerase; magnetic tweezers; nucleoid; single-molecule biophysics; stationary phase; stress response; transcription.

Figure 1.. Dps compacts the nucleoid in stationary-phase E. coli.

(A) Schematic of the structure of DNA in a wild-type cell during stationary phase. Dps condenses the cellular DNA. (B) In Δdps cells, Dps-mediated DNA compaction cannot occur. (C, D) Fluorescence images of the nucleoid from wild-type and Δdps cells stained with Hoechst 33258 (cyan) were superimposed onto phase-contrast images of the same cells (black on red) grown for (C) 24 hours or (D) 96 hours. (E) Ratios of nucleoid length to cell length, extracted from fluorescence images (n = 133 - 208 cells per condition). The error bars represent the estimate of the standard errors by bootstrapping. See also Figure S1.

Figure 2.. Dps has no influence on the transcriptome and mild influence on the proteome in stationary-phase E. coli cells.

(A) The relative amounts of total RNA and mRNA (mean ± SE).. (B) Differential expression analysis of RNA sequencing. For each gene, the mean expression in the wild-type strain is plotted against the corresponding value in the Δdps strain. Colors represent the fold-difference between the two strains. (C) The significance of the shift in mean expression for each mRNA species (as determined by the p or q value) is plotted against the fold change. (D) Total protein levels (mean ± SE). (E) Differential expression profile of SILAC analysis. For each protein, the mean abundance in the wild-type strain (y-axis) is plotted against the corresponding value in the Δdps strain (x-axis). Colors represent the fold-difference between the two strains. (F) The significance of the shift in mean expression for each protein species (as determined by the p or q value) is plotted against the fold change. See also Figures S2 and S3.

Figure 3.. Dps allows RNAP to bind to promoters but excludes KpnI restriction enzyme from its target site.

(A) Gel-shift analysis of Dps binding to linear promoter DNA fragments. The calculated K_D and Hill coefficients resulting from fits to the Hill equation are summarized in Table S1. (B) Transcription initiation from the recA promoter. (C) Dps-mediated protection from DNA digestion. The vertical dashed lines in (B) and (C) indicate the K_D of Dps for the different DNA templates shown in (A). The data in panels A-C are shown as mean ± SD from three biological replicates. (D) Wild-type, K8A, or K10A Dps proteins at 4 μM were bound to recA DNA, followed by incubation with or without KpnI. DNA:Dps complexes were dissociated by heparin. See also Figures S4 and S5.

Figure 4.. Multiplexed single-molecule transcription-elongation assay and dwell time analysis of RNAP dynamics.

(A) Schematics of the single-molecule in vitro transcriptional assay in the assisting force (AF) configuration, showing a single RNAP bound to a surface-attached DNA template in the presence of Dps. A magnetic bead was attached to the RNAP and exerted a constant force of 5 pN on the ternary complex. (B) The opposing force (OF) experimental configuration (C) Individual RNAP trajectories over time measured at 25 Hz via the change of the diffraction pattern of the attached magnetic bead (inset). The dashed rectangle depicts the trace region magnified in (D). Dwell times (τ_n) associated with advancing 10 nt were extracted from 1 Hz-filtered elongation traces (black line). Boundaries denoted by blue dashed lines. The error bars represent the estimate of the standard errors by bootstrapping. See also Figure S6.

Figure 5.. Dependence of transcription elongation dynamics on force and location of DNA:Dps complex.

(A) Dwell time distributions for assisting force (AF) trajectories in the presence (red) and the absence (black) of 1 μM Dps at 20°C. (B) Dwell time distributions resulting from the opposing force (OF) experiments in the presence (blue) and the absence (black) of 1 μM Dps at 20°C. (C) Comparison of extracted RNAP elongation rates k for AF and OF experimental distributions shown in (A, B), determined by Galton distribution fits with an upper boundary of 1 s. (D, E) Calculated transcription pause probabilities (per 10 nt) for short (SP, D) and long pauses (LP, E) for the experimental configurations shown in (A, B). The error bars represent the SD. Statistical results, dwell times, and number of trajectories measured are summarized in Table S2. See also Figure S7.

Figure 6.. RNAP transcribes through a fully condensed DNA:Dps complex.

(A) The experimental configuration was similar to the OF configuration (see also Figure 4B) but at lower exerted force (0.7 pN), which allows Dps to condense the entire DNA tether. (B) Bead position traces for stalled RNAP on a condensed DNA:Dps complex, before (upper panel) and after (lower panel) transcription restarted upon the addition of rNTPs. (C) Two representative time traces of active RNAP on a condensed DNA:Dps complex. Steep upward jumps in bead position (black) were accompanied by gradual downward displacements (red). (D) Comparative box plot of noise levels (standard deviation) measured in the absence (grey) and in the presence of Dps prior to (purple) or following rNTP addition (cyan). (E) Example trace with transient pulling to 8 pN (blue) every 400 s to determine the absolute RNAP position (red) along the DNA tether. (F) Distribution of average velocities from (E). The red line indicates a Gaussian fit. (G) Comparative box plot of average velocities determined from the transient pulling (E) and transcription experiments for AF and OF configurations in the presence and the absence of Dps (see Figure 5). The outer confidence intervals of the box plots represent the 1.5 interquartile range. See also Figure S6.

Figure 7.. Proposed model of DNA protection by Dps.

(A) In unstressed cells, Dps binds DNA transiently but is unable to condense the vast majority of the nucleoid. (B) Under conditions of high stress, dense complexes of Dps cover a large fraction of the nucleoid, creating phase-separated organelles. While RNAP can freely enter and diffuse inside these Dps complexes, other proteins are blocked from accessing the DNA.