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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.

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Schematic of the coupled transcription–translation–transertion system in rapidly growing Escherichia coli. Adapted from Bakshi et al. (2014a).
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Figure 1: Schematic of the coupled transcription–translation–transertion system in rapidly growing Escherichia coli. Adapted from Bakshi et al. (2014a).

Mentions: In E. coli, the chromosomal DNA occupies the region of space called the nucleoids (Kellenberger, 1991). During rapid growth, ribosomes are concentrated outside the nucleoids in ribosome-rich regions comprising the two polar end-caps, the space between nucleoid lobes, and the thin region proximal to the cytoplasmic membrane (Figure 1; Bakshi et al., 2012). The spatial extent of the nucleoids evidently arises from a balance of compacting and expanding forces (Woldringh et al., 1995; Zimmerman, 2006). Likely compacting forces include depletion-attraction of DNA arising from macromolecular crowding by myriad small proteins (Zimmerman and Murphy, 1996); conformational entropy of the confined DNA polymer, which causes the polymer to avoid walls (Mondal et al., 2011; Bakshi et al., 2014a); inter-strand coupling by DNA binding proteins such as H-NS (Dame, 2005; Wang et al., 2011); bending of DNA by IHF (Dame, 2005); and net supercoiling of the DNA by Gyrase and Topoisomerase I (Woldringh et al., 1995). The hypothesized primary expanding force is “transertion,” which is the simultaneous co-transcriptional translation and insertion of membrane proteins via the translocon machinery (Woldringh, 2002). Transertion implies the existence of DNA-RNAP-mRNA-ribosome-polypeptide-membrane “transertion chains” directly linking DNA to the membrane (Figure 1). A sufficient number of these chains would radially expand the overall nucleoid. The main evidence for transertion had been the dramatic contraction of the nucleoids on treatment with translation-halting drugs such as chloramphenicol (van Helvoort et al., 1996; Zimmerman, 2002). Treatment with transcription-halting drugs such as rifampicin should have the same effect, but this was not observed on the 30-min timescale studied (Fishov and Woldringh, 1999; Cabrera et al., 2009). Our recent time-dependent imaging study discovered nucleoid contraction on a 3-min timescale after rifampicin treatment, placing the transertion hypothesis on solid footing (Bakshi et al., 2014a).


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

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

Schematic of the coupled transcription–translation–transertion system in rapidly growing Escherichia coli. Adapted from Bakshi et al. (2014a).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Schematic of the coupled transcription–translation–transertion system in rapidly growing Escherichia coli. Adapted from Bakshi et al. (2014a).
Mentions: In E. coli, the chromosomal DNA occupies the region of space called the nucleoids (Kellenberger, 1991). During rapid growth, ribosomes are concentrated outside the nucleoids in ribosome-rich regions comprising the two polar end-caps, the space between nucleoid lobes, and the thin region proximal to the cytoplasmic membrane (Figure 1; Bakshi et al., 2012). The spatial extent of the nucleoids evidently arises from a balance of compacting and expanding forces (Woldringh et al., 1995; Zimmerman, 2006). Likely compacting forces include depletion-attraction of DNA arising from macromolecular crowding by myriad small proteins (Zimmerman and Murphy, 1996); conformational entropy of the confined DNA polymer, which causes the polymer to avoid walls (Mondal et al., 2011; Bakshi et al., 2014a); inter-strand coupling by DNA binding proteins such as H-NS (Dame, 2005; Wang et al., 2011); bending of DNA by IHF (Dame, 2005); and net supercoiling of the DNA by Gyrase and Topoisomerase I (Woldringh et al., 1995). The hypothesized primary expanding force is “transertion,” which is the simultaneous co-transcriptional translation and insertion of membrane proteins via the translocon machinery (Woldringh, 2002). Transertion implies the existence of DNA-RNAP-mRNA-ribosome-polypeptide-membrane “transertion chains” directly linking DNA to the membrane (Figure 1). A sufficient number of these chains would radially expand the overall nucleoid. The main evidence for transertion had been the dramatic contraction of the nucleoids on treatment with translation-halting drugs such as chloramphenicol (van Helvoort et al., 1996; Zimmerman, 2002). Treatment with transcription-halting drugs such as rifampicin should have the same effect, but this was not observed on the 30-min timescale studied (Fishov and Woldringh, 1999; Cabrera et al., 2009). Our recent time-dependent imaging study discovered nucleoid contraction on a 3-min timescale after rifampicin treatment, placing the transertion hypothesis on solid footing (Bakshi et al., 2014a).

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