Abstract
Messenger RNA molecules have been localized to different positions in cells and have been followed by time-lapse microscopy. We have used MS2-mVenus–labeled mRNA and single-particle tracking to obtain information on the dynamics of single-mRNA molecules in real time. Using single-molecule tracking, we show that several mRNA molecules visualized via two MS2-binding sites and MS2-mVenus expressed in Bacillus subtilis cells show free diffusion through the entire cell and constrained motion predominantly close to the cell membrane and at the polar regions of the cells. Because constrained motion of mRNAs likely reflects molecules complexed with ribosomes, our data support the idea that translation occurs at sites surrounding the nucleoids. Squared displacement analyses show the existence of at least two distinct populations of molecules with different diffusion constants or possibly of three populations, for example, freely mobile mRNAs, mRNAs in transition complexes, or in complex with polysomes. Diffusion constants between differently sized mRNAs did not differ dramatically and were much lower than that of cytosolic proteins. These data agree with the large size of mRNA molecules and suggest that, within the viscous cytoplasm, size variations do not translate into mobility differences. However, at observed diffusion constants, mRNA molecules would be able to reach all positions within cells in a frame of seconds. We did not observe strong differences in the location of confined motion for mRNAs encoding mostly soluble or membrane proteins, indicating that there is no strong bias for localization of membrane protein-encoding transcripts for the cell membrane.
Highlights
In the past, it has been a common model that transcription and translation occur at the same space and time in bacteria (Miller et al, 1970), because of the fact that, in general, bacteria are non-compartmentalized cells, unlike eukaryotes
A diffusion coefficients (DCs) of 0.55 μm2 s−1 is surprisingly low for a small protein fused to mVenus, but at the acquisition speed used, we do not believe that our analyses are hampered by technical limitations
Until recent developments of high and super resolution imaging methods, it was thought that transcription and translation in bacteria happen in a temporally and spatially coupled manner (Miller et al, 1970)
Summary
It has been a common model that transcription and translation occur at the same space and time in bacteria (Miller et al, 1970), because of the fact that, in general, bacteria are non-compartmentalized cells, unlike eukaryotes. Even though bacteria lack internal membrane systems similar to those found in eukaryotes, they possess a high degree of three-dimensional organization Model bacteria such as Escherichia coli and Bacillus subtilis show a compacted structure called the nucleoid, containing the chromosome, which occupies the central space of the cell, but is absent at the cell poles or in the middle of large cells prior to cell division (Fisher et al, 2013). In different bacterial species, transcription and translation can take place in close spatial proximity; that is, genes and ribosomes can be found at the same place, or largely separated, when the chromosome is organized as a nucleoid In the latter case, the question arises how RNA moves from its places of synthesis on the nucleoids to the cell poles, and further, if mRNA might be translated near sites where the encoded protein is used, for example, in case of cell division proteins, or membrane proteins that localize to the cell poles. Different models could be possible in different bacteria species
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