Mechanistic insights into the spatial organization of RNA polymerase proteins and the chromosome in E. coli cells

Abstract

Along the bacterial chromosome, regions called rrn operons contain genes that are transcribed into ribosomal RNA. These operons are among the most transcriptionally active sites in the genome. It has been observed in E. coli that RNA polymerase (RNAP), while binding to these genetic loci along the chromosome during transcription, forms dense clusters, leading to spatial colocalization of the operons within the cell. Recent experimental evidence suggests that liquid-liquid phase separation contributes to the formation of RNAP clusters, with the antitermination factor NusA playing a key role. We present a simulation model to investigate the mechanisms underlying the formation of these biomolecular condensates. We propose that mutual attraction between NusA proteins, which exhibit a miscibility gap at higher concentrations, drives condensate formation via a polymer-assisted condensation pathway, and we demonstrate how these condensates promote the colocalization of rrn operons. Our results reconcile seemingly disparate experimental observations of chromosomal organization reported in fluorescence-based imaging and Hi-C experiments.

Figures (7)

• Figure 1: Schematic illustration of the coarse-graining and our model: (a) Left: the rrn operon sequence (red strand) binds to RNAP (yellow) and 'bound' Nus proteins (blue); 10 of 42 (both Nus and RNAP) proteins are shown. Right: the operon is mapped onto an effective monomer in our model. RNAP acts as a linker connecting bound Nus proteins to the operon. The Nus proteins form a corona around the operon. (b) Sketch of the coarse-grained model of the chromosome inside the cylindrical E. coli cell. The free Nus proteins are shown in green display attractive interaction and are also attracted by the corona particles attached to the rrn operons.
• Figure 2: Bulk phase diagram of the Nus: We display the bulk phase diagram of free Nus in the absence of the chromosome-polymer and bound Nus. The yellow region denotes the parameter regime where phase separation occurs, and the violet region corresponds to the homogeneous phase. The red circle ($\epsilon_{n}/\mathrm{k_B T}=2.5$ and $\rho_{nus}=0.29\, \mathrm{\mu M}$) corresponds to the bulk state which we select for our simulations.
• Figure 3: Simulation snapshots and probability distribution of distance between neighbouring Nus: (a) Snapshots (radial and the longitudinal view) from our simulations are displayed corresponding to $\epsilon_n=2.5\, \mathrm{k_B T}$ and $N_{f}=400$. The chromosome monomers are shown in red and monomers corresponding to the rrn operons are shown in green (enlarged). Bound Nus beads are shown in blue and free Nus shown in cyan (enlarged). In (b) we display the probability distribution of the distance between between a free Nus bead and its nearest free Nus neighbour. We display the distributions for $\epsilon_n=2.5\, \mathrm{k_B T}$, $\epsilon_n=2.0\, \mathrm{k_B T}$ and when there are purely repulsive interactions between the Nus. The distribution has been computed with data collected after every $10,000$ MCS, from $1\times10^6$ MCS to $7.5\times 10^6$ MCS.
• Figure 4: Number of Nus particles in cluster and spatial distribution of components: In (a) we display the probability distribution of the number of free Nus particles in the largest cluster ($N_{cluster}$), formed during a simulation run. We display data for $\epsilon_n=2.5\, \mathrm{k_B T}$ and for the case when there are only repulsive interactions among Nus. In (b) we display the radial probability densities $p(r)$ of the various components as a function of the distance $r$ from the center of mass (COM) of the cluster. The quantity $p(r)\cdot4\pi r^{2}\,dr$ gives the probability of finding a particle in a spherical shell between $r$ and $r+dr$. The radial bin size used in the calculation is $\delta r=15\,\mathrm{nm}$. The distributions shown in (a) and (b) have been computed with data collected after every $10,000$ MCS from $1\times10^6$ MCS to $7.5\times 10^6$ MCS.
• Figure 5: Dynamics of Nus in the two phases: In (a) we display the probability distribution of free Nus to be inside the condensate for time $\tau$ (MCS). The mean residence time $\tau_r$, is extracted by fitting an exponential function: $\mathrm{P(0)}\cdot \exp(-\tau/\tau_r)$ to the curve. The mean residence time is $\tau_r \simeq 11,000$ MCS. In (b) we display the distributions of diffusion constants of free Nus in the dilute and the dense phase respectively. The most probable diffusion constant value in the two phases differ by approximately two orders of magnitude