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Hot off the Press: Physical Nature of Bacterial Cytoplasms

by PhilipJ on 13 March 2006

The interior of a cell, even for relatively simple bacterial systems, is crowded. David Goodsell work shows this in excellent detail—we can’t think of the interior of, say, E. coli (see our header), as being a simple, homogeneous liquid. And a paper in this week’s PRL (subscription required) confirms it.

By tagging an RNA molecule with a green fluorescent protein construct, a group from Princeton tracked its diffusion in real-time via light microscopy inside a living E. coli cell for many minutes. Their experiments looked like this:

which clearly shows a dominating bright signal diffusing around the whole cell. More detailed analysis of the particle’s position in time revealed that it had unique dynamics, made up of periods of highly localized motion separated by fast jumps to new positions in the cell.

In fact, for pure Brownian motion, the Einstein-Smoluchowski relation says the mean squared displacement of a diffusing particle will go as

2(τ)> = 6Dτ,

where D is the diffusion coefficient, and τ is time. Analysis of the diffusing RNA molecules revealed instead that <δ2(τ)> goes as τα, where α is approximately 0.7. In Brownian diffusion, the particles make random jumps in space due to thermal fluctuations, but in subdiffusive systems, characterised by α < 1, some types of random media can effectively "trap" particles in a specific location, with infrequent jumps about the cell. This could be temporal in origin, where the diffusing molecule may bind to obstacles in the cellular milieu. By modifying the RNA molecule to include ribosome binding sites, the diffusion was seen to decrease further due to interactions with the 104 or so ribosomes present in the cell.

Looking closer at the dynamics of a diffusion, for a regular Brownian particle, the time it takes to move a distance l scales as l2, but for a subdiffusive particle it scales as l2/α. Similarly, if the particle is a distance r away from a target of size a, it has a probability of finding the target given by (a/r)3-2/α. This effectively means that it will take a subdiffusive particle longer to find its target, but hang around the general area for longer than a regular, brownian particle.

This naturally leads into asking how transcription factors are able to find their binding sites along DNA. Transcription factors are normally present in extremely low copy number, from a few to a hundred, and must find a very particular site on the genomic DNA to bind and regulate gene expression. Two famous transcription factors, the Phage λ and Lac repressors, are coded immediately adjacent to their respective binding sites. The transcription factors themselves are probably translated less than a hundred nanometres away from their associated binding sites, and the subdiffusive character of the cytoplasm will give them the time required to find their binding sites and carry out regulation. Fascinating!

  1. samudrika    4028 days ago    #

    Why did they tag the GFP to RNA? Why not use GFP alone? Or is the RNA targeted somewhere.

    Maybe its in the article but I could not access that.

    p.s. I read this blog regularly. Keep up the good work!

  2. PhilipJ    4028 days ago    #

    Hi samudrika, thanks!

    I guess I didn’t make that very clear. :) Their cells had many copies of the GFP construct, and the RNA molecule was engineered such that it had 90 or so GFP binding sites, which makes it very easy to get the signal from a single RNA molecule from the background GFP noise (you can see there is a non-zero background in the images above, but the RNA molecule is quite easy to pick out!).

    Also, using RNA instead of something else made it possible to add the ribosome binding sites, to see if that had an effect on the diffusive properties. I think there are people doing single-GFP detection, but it is quite a bit harder to do.

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