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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) SYTOX orange-stained image of chromosomal DNA in a live E. coli cell growing in EZRDM at 30°C. The nucleoid spatial extent is characterized by length LDNA and width WDNA measured as the full width at half-maximum height (FWHM) of intensity distributions projected onto the x and y axes. (B) Time-lapse sequences of images of nucleoids stained by SYTOX Orange. Times in minutes, scale bars are 1 mm. Untreated cells, Rif-treated cells, and Cam-treated cells as indicated. (C) Quantitative nucleoid width WDNA vs. time. Gray: mean behavior of a set of untreated cells. Blue: behavior of Rif-treated cells. Red: behavior of Cam-treated cells. For blue and red, heavy lines are averages of traces from many cells; shaded regions show the envelope of single-cell results that were averaged. Dashed line shows time of initiation of flow of drug. (D) Same as (C), but with relative nucleoid length plotted as LDNA/Lcell. Adapted from Bakshi et al. (2014a).
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Figure 8: (A) SYTOX orange-stained image of chromosomal DNA in a live E. coli cell growing in EZRDM at 30°C. The nucleoid spatial extent is characterized by length LDNA and width WDNA measured as the full width at half-maximum height (FWHM) of intensity distributions projected onto the x and y axes. (B) Time-lapse sequences of images of nucleoids stained by SYTOX Orange. Times in minutes, scale bars are 1 mm. Untreated cells, Rif-treated cells, and Cam-treated cells as indicated. (C) Quantitative nucleoid width WDNA vs. time. Gray: mean behavior of a set of untreated cells. Blue: behavior of Rif-treated cells. Red: behavior of Cam-treated cells. For blue and red, heavy lines are averages of traces from many cells; shaded regions show the envelope of single-cell results that were averaged. Dashed line shows time of initiation of flow of drug. (D) Same as (C), but with relative nucleoid length plotted as LDNA/Lcell. Adapted from Bakshi et al. (2014a).

Mentions: It has long been known that transcription- and translation-halting drugs strongly affect nucleoid morphology. Typical imaging experiments have compared nucleoid morphology before and 20–30 min after drug treatment, usually using DAPI as the DNA stain. We used the non-perturbative DNA stain SYTOX Orange (Bakshi et al., 2014b) to study time-dependent, quantitative effects of Rif and Cam on nucleoid length and width in live cells over 20 min (Bakshi et al., 2014a). To describe the overall spatial distribution of the chromosomal DNA vs. time, we defined two parameters measured from the SYTOX Orange fluorescence intensity distributions projected along the x- and y-axes (Figure 8A). The axial distribution was characterized by the overall length LDNA, measured as the “outside” full-width at half-maximum height (FWHM) of the projected intensity distribution along x. The width WDNA, a rough measure of the mean nucleoid diameter, was defined as the FWHM of the projection of intensity along the transverse coordinate y. We believe this definition of nucleoid length and width is more quantitative than the “relative nucleoid size” used in other work (Jin et al., 2013). We also measured single-ribosome diffusive motion vs. time after drug treatment, using the 30S-mEos2 labeling scheme (Bakshi et al., 2014a). This helps distinguish 70S-polysomes from free 30S subunits after drug treatment.


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

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

(A) SYTOX orange-stained image of chromosomal DNA in a live E. coli cell growing in EZRDM at 30°C. The nucleoid spatial extent is characterized by length LDNA and width WDNA measured as the full width at half-maximum height (FWHM) of intensity distributions projected onto the x and y axes. (B) Time-lapse sequences of images of nucleoids stained by SYTOX Orange. Times in minutes, scale bars are 1 mm. Untreated cells, Rif-treated cells, and Cam-treated cells as indicated. (C) Quantitative nucleoid width WDNA vs. time. Gray: mean behavior of a set of untreated cells. Blue: behavior of Rif-treated cells. Red: behavior of Cam-treated cells. For blue and red, heavy lines are averages of traces from many cells; shaded regions show the envelope of single-cell results that were averaged. Dashed line shows time of initiation of flow of drug. (D) Same as (C), but with relative nucleoid length plotted as LDNA/Lcell. Adapted from Bakshi et al. (2014a).
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Figure 8: (A) SYTOX orange-stained image of chromosomal DNA in a live E. coli cell growing in EZRDM at 30°C. The nucleoid spatial extent is characterized by length LDNA and width WDNA measured as the full width at half-maximum height (FWHM) of intensity distributions projected onto the x and y axes. (B) Time-lapse sequences of images of nucleoids stained by SYTOX Orange. Times in minutes, scale bars are 1 mm. Untreated cells, Rif-treated cells, and Cam-treated cells as indicated. (C) Quantitative nucleoid width WDNA vs. time. Gray: mean behavior of a set of untreated cells. Blue: behavior of Rif-treated cells. Red: behavior of Cam-treated cells. For blue and red, heavy lines are averages of traces from many cells; shaded regions show the envelope of single-cell results that were averaged. Dashed line shows time of initiation of flow of drug. (D) Same as (C), but with relative nucleoid length plotted as LDNA/Lcell. Adapted from Bakshi et al. (2014a).
Mentions: It has long been known that transcription- and translation-halting drugs strongly affect nucleoid morphology. Typical imaging experiments have compared nucleoid morphology before and 20–30 min after drug treatment, usually using DAPI as the DNA stain. We used the non-perturbative DNA stain SYTOX Orange (Bakshi et al., 2014b) to study time-dependent, quantitative effects of Rif and Cam on nucleoid length and width in live cells over 20 min (Bakshi et al., 2014a). To describe the overall spatial distribution of the chromosomal DNA vs. time, we defined two parameters measured from the SYTOX Orange fluorescence intensity distributions projected along the x- and y-axes (Figure 8A). The axial distribution was characterized by the overall length LDNA, measured as the “outside” full-width at half-maximum height (FWHM) of the projected intensity distribution along x. The width WDNA, a rough measure of the mean nucleoid diameter, was defined as the FWHM of the projection of intensity along the transverse coordinate y. We believe this definition of nucleoid length and width is more quantitative than the “relative nucleoid size” used in other work (Jin et al., 2013). We also measured single-ribosome diffusive motion vs. time after drug treatment, using the 30S-mEos2 labeling scheme (Bakshi et al., 2014a). This helps distinguish 70S-polysomes from free 30S subunits after drug treatment.

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