DNA and mRNA as molecular speed bumps in Escherichia coli’s cytoplasm

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The cytosol of Escherichia coli is packed with ribosomes, DNA, mRNA, and proteins. If they were evenly distributed, the average distance between two biomacromolecules would be less than the size of a protein in E. coli. This proximity of surfaces means that biomacromolecules interact more frequently with each other, thereby enhancing attractive and repulsive weak interactions. Enhanced interactions induce organization and change reaction rates (1).

A prominent way to interrogate the resulting intracellular organization is by measuring the diffusion of a test molecule. Diffusion is a “passive rheology” experiment and is reduced by macromolecular crowding (excluded volume), as well as stickiness, immobile barriers (such as confinement, sieving effects, or obstructions), and hydrodynamic effects. Passive diffusion, which usually follows Brownian motion, is intrinsically particle-size dependent, and small-molecule and protein diffusion has been investigated in detail in cells. In recent years, the measurement of larger >20-nm structures, i.e., at the mesoscale length scale, has gained attention. The investigation of the mesoscale gained traction after the observation from the Jacobs-Wagner lab that the bacterial cytoplasm undergoes a transition to a glassy state upon energy depletion for such particles (2) In this glassy state, these larger particles, but not the smaller protein-sized ones, temporally stall. Various diffusion measurements at the mesoscale have shown that a range of cell stresses could alter nanoparticle diffusion through, e.g., crowding changes, ribosome concentration, mRNA content, nucleoid properties, or cytosol acidification (3,4,5).

It remains unclear what molecules determine intracellular mesoscale diffusion. The contribution of each molecule is challenging to determine, as each class of molecules has an organizational role in the cell, providing a mesoscale structure. Each of the most common classes of biomacromolecules (DNA, mRNA, ribosome, protein, glycogen) has been shown to cause a diffusion change. The challenge lies in determining how much each class of molecules contributes to the mesoscale diffusion change. Hence, directly comparing the main biomacromolecules would give valuable insight into which molecules dominate the mesoscale diffusion coefficients.

Writing in Biophysical Journal, Losa and Heinemann shed light on this issue by measuring the contribution of the different biomacromolecules to the diffusion of a 40-nm nanoparticle in E. coli (6). They employ a genetically encoded viral nanoparticle labeled with fluorescent proteins, which the Holt group previously used for diffusion measurements in yeast (3). The size of these particles allows single-particle tracking, where the average step size between images is used to estimate the diffusion coefficients. They first showed that different growth rates did not result in significant changes in diffusion. To test the effect of mRNA on nanoparticle diffusion, the authors inhibited transcription with rifampicin. The mRNA lifetime in E. coli is short, so inhibiting mRNA synthesis is an effective way to deplete the cell from it. While mRNA was estimated to constitute only <1% cell dry weight, it induced a significant increase in diffusion when it was removed. To test the effect of DNA, the authors expressed Isce-I with arabinose. This enzyme cleaves DNA, resulting in further digestion by exonucleases. Interestingly, the diffusion of the nanoparticle increased: this again shows the difference with a smaller inert protein diffusion, as this was previously shown to be unaffected by chromosome digestion (7). Moreover, it was shown that DNA removal increased diffusion more than mRNA removal. The authors hypothesized that the removal of DNA would also deplete the cells of mRNA. They elegantly tested this hypothesis by adding rifampicin after DNA digestion. Indeed, as expected, rifampicin did not affect diffusion after DNA digestion, indicating that the higher diffusion change lines up with combined mRNA and DNA removal. Even though the diffusion after DNA and mRNA digestion was faster, it was still not the same as in the buffer. This means that while DNA contributes ∼16% and mRNA ∼22%, the remainder, including proteins and ribosomes, still contributes to ∼63% to the diffusion observed.

By determining the excluded volume, the authors provide more insight into the relative obstruction of the nanoparticle. The excluded volume contrasts with the volume fraction in that the former is the volume a particular test particle cannot access and is dependent on the size of the test particle, while the latter is the volume occupied by the macromolecules. Using a simple mathematical model, the authors calculated an excluded volume from the relative reduction in diffusion compared to a dilute solution. By assuming the excluded volumes were additive, the contribution of each component could be assessed. Following such analysis, the excluded volume of DNA and mRNA would be 10% of the cell volume, while their volume fractions are 1.4% and 0.3%, respectively. Moreover, a staggering 80% of the cell volume would be inaccessible to the 40-nm particle in this analysis. While the authors studied this 40-nm particle, it would be interesting to see if more cell volume would become available and eventually approach the biopolymer volume fraction if smaller test particles were measured.

The authors speculate that the DNA influences the diffusion through its meshwork. Usually, a pore size of 50 nm is estimated for DNA, but this is an average value, and some parts may be less penetrable for the 40-nm particle. mRNA is much smaller than DNA, and its role in diffusion is more challenging to explain. It may be that rifampicin also depletes some ribosomal RNA, but mRNA depletion will also affect larger mRNA structures such as polyribosomes and transertions, during which an mRNA encoding a membrane protein is synthesized and translated at the membrane. Large reorganizations can thus be induced in the cytoplasm by removing RNA and DNA. 60% of the diffusion is in the hands of the proteins and ribosomes, which aligns with previous work that showed that ribosomes control larger particle diffusion in yeast.

The impact of the work lies in a deeper understanding of how much the different biomacromolecules influence the biochemical organization. This quantification is a reference work that allows the generation of well-informed hypotheses to explain why certain stress conditions change nanoparticle diffusion. For example, one could extrapolate that altering the shape of DNA will drastically affect nanoparticle diffusion. Indeed, cell wall damage drastically reduces diffusion (8), which is concomitant with an expansion of the nucleoid (9).

These findings will help future research, such as determining whether mesoscale diffusion impacts cell behavior and organization, which includes cytosolic dynamics and biomolecular phase separation. When the organization at the mesoscale is better understood, we can appreciate its functional relevance and universality and predict when cells need to adapt their organization. For example, a deeper insight into the size dependence of the mesoscale test particle on the cellular components will teach us what sizes of biochemical complexes can be affected and when their reactions are diffusion limited and need to be countered by increasing the substrate concentration. Moreover, this allows assessing the transition between the protein and the mesoscale, giving insight into organizational properties such as sieving and when these exciting mesoscale effects may become essential for a cell.

Editor: Guy Genin.