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The spatial biology of transcription and translation in rapidly growing Escherichia coli.

Bakshi S, Choi H, Weisshaar JC - Front Microbiol (2015)

Bottom Line: Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone.There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain.Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular Biophysics Program, University of Wisconsin-Madison, Madison WI, USA.

ABSTRACT
Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0-3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20-30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites.

No MeSH data available.


Related in: MedlinePlus

Schematic of the super-resolution imaging method. (A) Each fluorescent molecule makes a diffraction-limited, essentially Gaussian image on the camera. A dense set of normal labels (blue images) makes overlapping images. In each imaging cycle, a sparse set of labels is photoactivated (red image) and localized from the well-isolated, single-molecule images. (B) An image of the spatial distribution is built up one molecule at a time over 100s or 1000s of optical cycles (upper “half-cells”). Successive images of the same molecule form a diffusive trajectory (lower “half-cells”).
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Figure 2: Schematic of the super-resolution imaging method. (A) Each fluorescent molecule makes a diffraction-limited, essentially Gaussian image on the camera. A dense set of normal labels (blue images) makes overlapping images. In each imaging cycle, a sparse set of labels is photoactivated (red image) and localized from the well-isolated, single-molecule images. (B) An image of the spatial distribution is built up one molecule at a time over 100s or 1000s of optical cycles (upper “half-cells”). Successive images of the same molecule form a diffusive trajectory (lower “half-cells”).

Mentions: Super-resolution fluorescence microscopy of specific proteins in live cells (Figure 2) was enabled by three key technical advances. First, genetic manipulations enable replacement of the gene for the target protein by a gene that appends a fluorescent protein to the target. This “GFP revolution” enables imaging of a specific target protein in a live cell (Zhang et al., 2002). But caution is needed. The fluorescent protein tags may affect the function, aggregation state, spatial distribution, or movement of the target protein (Landgraf et al., 2012).


The spatial biology of transcription and translation in rapidly growing Escherichia coli.

Bakshi S, Choi H, Weisshaar JC - Front Microbiol (2015)

Schematic of the super-resolution imaging method. (A) Each fluorescent molecule makes a diffraction-limited, essentially Gaussian image on the camera. A dense set of normal labels (blue images) makes overlapping images. In each imaging cycle, a sparse set of labels is photoactivated (red image) and localized from the well-isolated, single-molecule images. (B) An image of the spatial distribution is built up one molecule at a time over 100s or 1000s of optical cycles (upper “half-cells”). Successive images of the same molecule form a diffusive trajectory (lower “half-cells”).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488752&req=5

Figure 2: Schematic of the super-resolution imaging method. (A) Each fluorescent molecule makes a diffraction-limited, essentially Gaussian image on the camera. A dense set of normal labels (blue images) makes overlapping images. In each imaging cycle, a sparse set of labels is photoactivated (red image) and localized from the well-isolated, single-molecule images. (B) An image of the spatial distribution is built up one molecule at a time over 100s or 1000s of optical cycles (upper “half-cells”). Successive images of the same molecule form a diffusive trajectory (lower “half-cells”).
Mentions: Super-resolution fluorescence microscopy of specific proteins in live cells (Figure 2) was enabled by three key technical advances. First, genetic manipulations enable replacement of the gene for the target protein by a gene that appends a fluorescent protein to the target. This “GFP revolution” enables imaging of a specific target protein in a live cell (Zhang et al., 2002). But caution is needed. The fluorescent protein tags may affect the function, aggregation state, spatial distribution, or movement of the target protein (Landgraf et al., 2012).

Bottom Line: Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone.There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain.Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Molecular Biophysics Program, University of Wisconsin-Madison, Madison WI, USA.

ABSTRACT
Single-molecule fluorescence provides high resolution spatial distributions of ribosomes and RNA polymerase (RNAP) in live, rapidly growing Escherichia coli. Ribosomes are more strongly segregated from the nucleoids (chromosomal DNA) than previous widefield fluorescence studies suggested. While most transcription may be co-translational, the evidence indicates that most translation occurs on free mRNA copies that have diffused from the nucleoids to a ribosome-rich region. Analysis of time-resolved images of the nucleoid spatial distribution after treatment with the transcription-halting drug rifampicin and the translation-halting drug chloramphenicol shows that both drugs cause nucleoid contraction on the 0-3 min timescale. This is consistent with the transertion hypothesis. We suggest that the longer-term (20-30 min) nucleoid expansion after Rif treatment arises from conversion of 70S-polysomes to 30S and 50S subunits, which readily penetrate the nucleoids. Monte Carlo simulations of a polymer bead model built to mimic the chromosomal DNA and ribosomes (either 70S-polysomes or 30S and 50S subunits) explain spatial segregation or mixing of ribosomes and nucleoids in terms of excluded volume and entropic effects alone. A comprehensive model of the transcription-translation-transertion system incorporates this new information about the spatial organization of the E. coli cytoplasm. We propose that transertion, which radially expands the nucleoids, is essential for recycling of 30S and 50S subunits from ribosome-rich regions back into the nucleoids. There they initiate co-transcriptional translation, which is an important mechanism for maintaining RNAP forward progress and protecting the nascent mRNA chain. Segregation of 70S-polysomes from the nucleoid may facilitate rapid growth by shortening the search time for ribosomes to find free mRNA concentrated outside the nucleoid and the search time for RNAP concentrated within the nucleoid to find transcription initiation sites.

No MeSH data available.


Related in: MedlinePlus