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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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Average spatial distribution of lacI-venus expression. Cells were grown in M63 minimal medium+0.5% glycerol before fixation. Averages are for cells of 1.9–2.2 μm in length, the bin identified in Figure 1D. Averages are also taken over all cell orientations, resulting in the symmetry of each plot. Raw images of combined transmitted and fluorescence channels are shown in the first column for DNA ( × 400, DAPI staining; row 1), gene locus ( × 400, FROS; row 2), mRNA ( × 400, FISH; row 3), LacI-Venus protein ( × 400, TIRF microscopy, row 4), and LacI-Venus protein without DNA binding domain ( × 400, TIRF microscopy, row 5). Representative LacI-Venus images are at × 133magnification for visual clarity. Remaining columns show average distributions for each component with the lacI-venus source gene expressed from the indicated location. Fluorescent intensity is normalized to the mean fluorescence within the cell. The quantitative color scale is for LacI-Venus only; other averages are qualitatively scaled for comparison. The essQ locus was the LacI-Venus source for terminus, but the gene location shown is for the adjacent nth terminal locus for clarity (Kuhlman and Cox, 2010); essQ localization is identical but less clear due to reduced binding at that locus.
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f2: Average spatial distribution of lacI-venus expression. Cells were grown in M63 minimal medium+0.5% glycerol before fixation. Averages are for cells of 1.9–2.2 μm in length, the bin identified in Figure 1D. Averages are also taken over all cell orientations, resulting in the symmetry of each plot. Raw images of combined transmitted and fluorescence channels are shown in the first column for DNA ( × 400, DAPI staining; row 1), gene locus ( × 400, FROS; row 2), mRNA ( × 400, FISH; row 3), LacI-Venus protein ( × 400, TIRF microscopy, row 4), and LacI-Venus protein without DNA binding domain ( × 400, TIRF microscopy, row 5). Representative LacI-Venus images are at × 133magnification for visual clarity. Remaining columns show average distributions for each component with the lacI-venus source gene expressed from the indicated location. Fluorescent intensity is normalized to the mean fluorescence within the cell. The quantitative color scale is for LacI-Venus only; other averages are qualitatively scaled for comparison. The essQ locus was the LacI-Venus source for terminus, but the gene location shown is for the adjacent nth terminal locus for clarity (Kuhlman and Cox, 2010); essQ localization is identical but less clear due to reduced binding at that locus.

Mentions: The average distributions of DNA, lacI-venus gene location, lacI-venus mRNA, and LacI-Venus protein for cells that have just divided are shown in Figure 2. We find that the replication origin-proximal integration locus is located midcell and the terminus-proximal locus at a superposition of the poles and midcell (Bates and Kleckner, 2005; Wiggins et al, 2010). These results are in broad agreement with the existing literature (Niki et al, 2000; Wang et al, 2006; Montero Llopis et al, 2010; Wiggins et al, 2010). FISH reveals lacI mRNA to be localized at the gene loci for origin and terminus integrants (Montero Llopis et al, 2010). When the lacI source gene is located on a medium copy number plasmid localized to the poles (Figure 2, second column), lacI-venus RNA shows some tendency to redistribute to other locations in the cell and LacI-Venus is evenly distributed throughout the nucleoid. When the source is located within the chromosome, however, we find that the distributions depend on the position of the encoding gene on the chromosome, and correlate with the spatial distributions of the source genes.


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

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

Average spatial distribution of lacI-venus expression. Cells were grown in M63 minimal medium+0.5% glycerol before fixation. Averages are for cells of 1.9–2.2 μm in length, the bin identified in Figure 1D. Averages are also taken over all cell orientations, resulting in the symmetry of each plot. Raw images of combined transmitted and fluorescence channels are shown in the first column for DNA ( × 400, DAPI staining; row 1), gene locus ( × 400, FROS; row 2), mRNA ( × 400, FISH; row 3), LacI-Venus protein ( × 400, TIRF microscopy, row 4), and LacI-Venus protein without DNA binding domain ( × 400, TIRF microscopy, row 5). Representative LacI-Venus images are at × 133magnification for visual clarity. Remaining columns show average distributions for each component with the lacI-venus source gene expressed from the indicated location. Fluorescent intensity is normalized to the mean fluorescence within the cell. The quantitative color scale is for LacI-Venus only; other averages are qualitatively scaled for comparison. The essQ locus was the LacI-Venus source for terminus, but the gene location shown is for the adjacent nth terminal locus for clarity (Kuhlman and Cox, 2010); essQ localization is identical but less clear due to reduced binding at that locus.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Average spatial distribution of lacI-venus expression. Cells were grown in M63 minimal medium+0.5% glycerol before fixation. Averages are for cells of 1.9–2.2 μm in length, the bin identified in Figure 1D. Averages are also taken over all cell orientations, resulting in the symmetry of each plot. Raw images of combined transmitted and fluorescence channels are shown in the first column for DNA ( × 400, DAPI staining; row 1), gene locus ( × 400, FROS; row 2), mRNA ( × 400, FISH; row 3), LacI-Venus protein ( × 400, TIRF microscopy, row 4), and LacI-Venus protein without DNA binding domain ( × 400, TIRF microscopy, row 5). Representative LacI-Venus images are at × 133magnification for visual clarity. Remaining columns show average distributions for each component with the lacI-venus source gene expressed from the indicated location. Fluorescent intensity is normalized to the mean fluorescence within the cell. The quantitative color scale is for LacI-Venus only; other averages are qualitatively scaled for comparison. The essQ locus was the LacI-Venus source for terminus, but the gene location shown is for the adjacent nth terminal locus for clarity (Kuhlman and Cox, 2010); essQ localization is identical but less clear due to reduced binding at that locus.
Mentions: The average distributions of DNA, lacI-venus gene location, lacI-venus mRNA, and LacI-Venus protein for cells that have just divided are shown in Figure 2. We find that the replication origin-proximal integration locus is located midcell and the terminus-proximal locus at a superposition of the poles and midcell (Bates and Kleckner, 2005; Wiggins et al, 2010). These results are in broad agreement with the existing literature (Niki et al, 2000; Wang et al, 2006; Montero Llopis et al, 2010; Wiggins et al, 2010). FISH reveals lacI mRNA to be localized at the gene loci for origin and terminus integrants (Montero Llopis et al, 2010). When the lacI source gene is located on a medium copy number plasmid localized to the poles (Figure 2, second column), lacI-venus RNA shows some tendency to redistribute to other locations in the cell and LacI-Venus is evenly distributed throughout the nucleoid. When the source is located within the chromosome, however, we find that the distributions depend on the position of the encoding gene on the chromosome, and correlate with the spatial distributions of the source genes.

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