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Gene location and DNA density determine transcription factor distributions in Escherichia coli.

Kuhlman TE, Cox EC - Mol. Syst. Biol. (2012)

Bottom Line: Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene.We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid.We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA. tkuhlman@illinois.edu

ABSTRACT
The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

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LacI redistribution as a result of nuceloid condensation. (A) The steady-state distribution of DNA and LacI-Venus in cells with lacI-venus integrated near the origin (atpI locus) and grown in M63+0.5% glycerol, cell length of 4.2–4.5 μm, are shown at t=0. After this sample was taken, 200 μg/ml chloramphenicol was added directly to the culture and samples were withdrawn and fixed at the indicated times. Note that the color scale has been changed somewhat from Figure 4 to accommodate the condensed DNA. (B) Cross-sections along the longitudinal axis of the DNA (blue) and LacI-Venus (red) distributions at time t=0 (top) and t=10 min (bottom). Lines are a moving average of six adjacent points. (C) The rate of mass displacement. Displacement is quantified as the total difference between each distribution and the initial distribution at time t=0. The black line corresponds to a simultaneous fit of both data sets to the equation . The equivalent result for LacI42-Venus is shown in Supplementary Figure 6. Source data is available for this figure in the Supplementary Information.
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f5: LacI redistribution as a result of nuceloid condensation. (A) The steady-state distribution of DNA and LacI-Venus in cells with lacI-venus integrated near the origin (atpI locus) and grown in M63+0.5% glycerol, cell length of 4.2–4.5 μm, are shown at t=0. After this sample was taken, 200 μg/ml chloramphenicol was added directly to the culture and samples were withdrawn and fixed at the indicated times. Note that the color scale has been changed somewhat from Figure 4 to accommodate the condensed DNA. (B) Cross-sections along the longitudinal axis of the DNA (blue) and LacI-Venus (red) distributions at time t=0 (top) and t=10 min (bottom). Lines are a moving average of six adjacent points. (C) The rate of mass displacement. Displacement is quantified as the total difference between each distribution and the initial distribution at time t=0. The black line corresponds to a simultaneous fit of both data sets to the equation . The equivalent result for LacI42-Venus is shown in Supplementary Figure 6. Source data is available for this figure in the Supplementary Information.

Mentions: Before replication, the origin locus produces LacI-Venus at midcell. After replication and segregation, LacI-Venus pools in the inter-chromatid space near where the origin was originally, as judged by Figures 4 and 5. Similarly, after the terminus relocates to midcell after replication and segregation, there remains a pool of LacI-Venus further out toward the ends of the cell. We propose that this tendency of LacI-Venus to pool in the nearest available space that contains less DNA is also a result of a tendency toward nucleoid exclusion of LacI-Venus molecules, and may be related to previously observed asymmetry of the cell poles (Rang Camilla et al, 2011).


Gene location and DNA density determine transcription factor distributions in Escherichia coli.

Kuhlman TE, Cox EC - Mol. Syst. Biol. (2012)

LacI redistribution as a result of nuceloid condensation. (A) The steady-state distribution of DNA and LacI-Venus in cells with lacI-venus integrated near the origin (atpI locus) and grown in M63+0.5% glycerol, cell length of 4.2–4.5 μm, are shown at t=0. After this sample was taken, 200 μg/ml chloramphenicol was added directly to the culture and samples were withdrawn and fixed at the indicated times. Note that the color scale has been changed somewhat from Figure 4 to accommodate the condensed DNA. (B) Cross-sections along the longitudinal axis of the DNA (blue) and LacI-Venus (red) distributions at time t=0 (top) and t=10 min (bottom). Lines are a moving average of six adjacent points. (C) The rate of mass displacement. Displacement is quantified as the total difference between each distribution and the initial distribution at time t=0. The black line corresponds to a simultaneous fit of both data sets to the equation . The equivalent result for LacI42-Venus is shown in Supplementary Figure 6. Source data is available for this figure in the Supplementary Information.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3472691&req=5

f5: LacI redistribution as a result of nuceloid condensation. (A) The steady-state distribution of DNA and LacI-Venus in cells with lacI-venus integrated near the origin (atpI locus) and grown in M63+0.5% glycerol, cell length of 4.2–4.5 μm, are shown at t=0. After this sample was taken, 200 μg/ml chloramphenicol was added directly to the culture and samples were withdrawn and fixed at the indicated times. Note that the color scale has been changed somewhat from Figure 4 to accommodate the condensed DNA. (B) Cross-sections along the longitudinal axis of the DNA (blue) and LacI-Venus (red) distributions at time t=0 (top) and t=10 min (bottom). Lines are a moving average of six adjacent points. (C) The rate of mass displacement. Displacement is quantified as the total difference between each distribution and the initial distribution at time t=0. The black line corresponds to a simultaneous fit of both data sets to the equation . The equivalent result for LacI42-Venus is shown in Supplementary Figure 6. Source data is available for this figure in the Supplementary Information.
Mentions: Before replication, the origin locus produces LacI-Venus at midcell. After replication and segregation, LacI-Venus pools in the inter-chromatid space near where the origin was originally, as judged by Figures 4 and 5. Similarly, after the terminus relocates to midcell after replication and segregation, there remains a pool of LacI-Venus further out toward the ends of the cell. We propose that this tendency of LacI-Venus to pool in the nearest available space that contains less DNA is also a result of a tendency toward nucleoid exclusion of LacI-Venus molecules, and may be related to previously observed asymmetry of the cell poles (Rang Camilla et al, 2011).

Bottom Line: Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene.We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid.We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Princeton University, Princeton, NJ, USA. tkuhlman@illinois.edu

ABSTRACT
The diffusion coefficient of the transcription factor LacI within living Escherichia coli has been measured directly by in vivo tracking to be D = 0.4 μm(2)/s. At this rate, simple models of diffusion lead to the expectation that LacI and other proteins will rapidly homogenize throughout the cell. Here, we test this expectation of spatial homogeneity by single-molecule visualization of LacI molecules non-specifically bound to DNA in fixed cells. Contrary to expectation, we find that the distribution depends on the spatial location of its encoding gene. We demonstrate that the spatial distribution of LacI is also determined by the local state of DNA compaction, and that E. coli can dynamically redistribute proteins by modifying the state of its nucleoid. Finally, we show that LacI inhomogeneity increases the strength with which targets located proximally to the LacI gene are regulated. We propose a model for intranucleoid diffusion that can reconcile these results with previous measurements of LacI diffusion, and we discuss the implications of these findings for gene regulation in bacteria and eukaryotes.

Show MeSH
Related in: MedlinePlus