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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

(A) Hyper-branched polymer bead model of plectonemic DNA and 70S-polysomes. The red DNA beads exclude each other; the gray beads are invisible to the DNA beads but act as volume appropriately excluded to the polysomes. Polysomes are represented as freely jointed chains of spheres of appropriate size. (B) In Monte Carlo simulations, 70S-polysomes and DNA strongly avoid each other. (C) When the 70S-polysomes are dissociated into 50S and 30S monomers, the simulations show strong mixing and nucleoid expansion. Adapted from Bakshi et al. (2014a).
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Figure 10: (A) Hyper-branched polymer bead model of plectonemic DNA and 70S-polysomes. The red DNA beads exclude each other; the gray beads are invisible to the DNA beads but act as volume appropriately excluded to the polysomes. Polysomes are represented as freely jointed chains of spheres of appropriate size. (B) In Monte Carlo simulations, 70S-polysomes and DNA strongly avoid each other. (C) When the 70S-polysomes are dissociated into 50S and 30S monomers, the simulations show strong mixing and nucleoid expansion. Adapted from Bakshi et al. (2014a).

Mentions: In Mondal et al. (2011), we developed a simple physical model of plectonemic DNA and 70S-polysomes confined in a spherocylinder (as pictured in Figure 10A). DNA was modeled as a hyperbranched polymer (hard spheres and connecting rods). Based on estimates at the time (Bremer and Dennis, 1996), the model placed two chromosome equivalents of plectonemic DNA (comprising 7000 plectoneme rods) plus 20,000 70S particles organized as freely jointed 70S-polysome 13-mers into a spherocylinder (350 nm radius, 3.0 μm length). DNA–DNA, polysome–DNA, and polysome–polysome excluded volume effects were modeled realistically. There are no attractive interactions between particles. The free energy of the composite system was minimized using Monte Carlo methods. This minimalist, coarse-grained model does not include the effects of transertion, nor does it attempt to describe the level of geometric detail revealed by recent chromosome conformational capture data (Cagliero et al., 2013; Le et al., 2013; Le and Laub, 2014). Instead, it seeks to understand the effects of excluded volume and entropy on the overall spatial distributions of DNA and ribosomes.


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

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

(A) Hyper-branched polymer bead model of plectonemic DNA and 70S-polysomes. The red DNA beads exclude each other; the gray beads are invisible to the DNA beads but act as volume appropriately excluded to the polysomes. Polysomes are represented as freely jointed chains of spheres of appropriate size. (B) In Monte Carlo simulations, 70S-polysomes and DNA strongly avoid each other. (C) When the 70S-polysomes are dissociated into 50S and 30S monomers, the simulations show strong mixing and nucleoid expansion. Adapted from Bakshi et al. (2014a).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: (A) Hyper-branched polymer bead model of plectonemic DNA and 70S-polysomes. The red DNA beads exclude each other; the gray beads are invisible to the DNA beads but act as volume appropriately excluded to the polysomes. Polysomes are represented as freely jointed chains of spheres of appropriate size. (B) In Monte Carlo simulations, 70S-polysomes and DNA strongly avoid each other. (C) When the 70S-polysomes are dissociated into 50S and 30S monomers, the simulations show strong mixing and nucleoid expansion. Adapted from Bakshi et al. (2014a).
Mentions: In Mondal et al. (2011), we developed a simple physical model of plectonemic DNA and 70S-polysomes confined in a spherocylinder (as pictured in Figure 10A). DNA was modeled as a hyperbranched polymer (hard spheres and connecting rods). Based on estimates at the time (Bremer and Dennis, 1996), the model placed two chromosome equivalents of plectonemic DNA (comprising 7000 plectoneme rods) plus 20,000 70S particles organized as freely jointed 70S-polysome 13-mers into a spherocylinder (350 nm radius, 3.0 μm length). DNA–DNA, polysome–DNA, and polysome–polysome excluded volume effects were modeled realistically. There are no attractive interactions between particles. The free energy of the composite system was minimized using Monte Carlo methods. This minimalist, coarse-grained model does not include the effects of transertion, nor does it attempt to describe the level of geometric detail revealed by recent chromosome conformational capture data (Cagliero et al., 2013; Le et al., 2013; Le and Laub, 2014). Instead, it seeks to understand the effects of excluded volume and entropy on the overall spatial distributions of DNA and ribosomes.

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