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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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Repression strength as a function of intergenic distance. Repression strength as a function of genomic distance from the lacI-venus gene, where lacI-venus is near (A) origin (atpI locus), (B) mid-replichore (ybbD locus), or (C) terminus (nth locus). Red: slow growth (τ=110±12 min); yellow: medium growth (τ=68±5 min); green: fast growth (τ=22±2 min); black: M63+0.5% glycerol+2 mM IPTG control; circles are the mean of four measurements, error bars are the s.d. Dashed lines indicate best fits assuming spatially homogeneous distribution of repressor. Solid lines indicate best fits assuming exponential LacI-Venus inhomogeneity. Source data is available for this figure in the Supplementary Information.
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f6: Repression strength as a function of intergenic distance. Repression strength as a function of genomic distance from the lacI-venus gene, where lacI-venus is near (A) origin (atpI locus), (B) mid-replichore (ybbD locus), or (C) terminus (nth locus). Red: slow growth (τ=110±12 min); yellow: medium growth (τ=68±5 min); green: fast growth (τ=22±2 min); black: M63+0.5% glycerol+2 mM IPTG control; circles are the mean of four measurements, error bars are the s.d. Dashed lines indicate best fits assuming spatially homogeneous distribution of repressor. Solid lines indicate best fits assuming exponential LacI-Venus inhomogeneity. Source data is available for this figure in the Supplementary Information.

Mentions: Because the strength with which bacterial promoters are regulated is a function of the concentration of the relevant transcription factors (Buchler et al, 2003; Bintu et al, 2005a, 2005b), we next attempt to detect the local enhancement of LacI concentration near the lacI gene through the repression strength of a LacI-regulated target. We constructed three families of 14 strains where we inserted into previously constructed lacI-venus strains a cassette bearing a LacI-regulated lacZ target at regular intervals around the genome (Supplementary Figure 1). In Figure 6A–C, the strength of repression is shown as a function of intergenic distance for each family of strains. Colors indicate the growth rate of the culture: slow (red), medium (yellow); fast (green); slow +2 mM IPTG control (black). In each plot, the x axis is adjusted so that the location of the repressor gene lies at the center of the x axis (black vertical dashed lines). In all cases, the strength of repression increases as the target is moved toward the terminus and decreases as the target is moved toward the origin. In each case there is a small but reproducible 1.2–2 × spike in repression when the target is located immediately proximal (∼200 bp downstream) to the repressor gene. We encounter little interference from position-specific effects, with a few exceptions: in all cases, the target inserted near the terminus at the essQ locus (see Supplementary Figure 1) remained unregulated. Additionally, when lacI-venus is inserted near the origin (Figure 6A) the repression strengths at essQ and nth near the terminus are significantly weaker than might otherwise be expected. This may be due to their location within the less densely packed chromosomal crossing region (Wiggins et al, 2010). These data were excluded from the best fit lines in Figure 6A–C.


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

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

Repression strength as a function of intergenic distance. Repression strength as a function of genomic distance from the lacI-venus gene, where lacI-venus is near (A) origin (atpI locus), (B) mid-replichore (ybbD locus), or (C) terminus (nth locus). Red: slow growth (τ=110±12 min); yellow: medium growth (τ=68±5 min); green: fast growth (τ=22±2 min); black: M63+0.5% glycerol+2 mM IPTG control; circles are the mean of four measurements, error bars are the s.d. Dashed lines indicate best fits assuming spatially homogeneous distribution of repressor. Solid lines indicate best fits assuming exponential LacI-Venus inhomogeneity. 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

f6: Repression strength as a function of intergenic distance. Repression strength as a function of genomic distance from the lacI-venus gene, where lacI-venus is near (A) origin (atpI locus), (B) mid-replichore (ybbD locus), or (C) terminus (nth locus). Red: slow growth (τ=110±12 min); yellow: medium growth (τ=68±5 min); green: fast growth (τ=22±2 min); black: M63+0.5% glycerol+2 mM IPTG control; circles are the mean of four measurements, error bars are the s.d. Dashed lines indicate best fits assuming spatially homogeneous distribution of repressor. Solid lines indicate best fits assuming exponential LacI-Venus inhomogeneity. Source data is available for this figure in the Supplementary Information.
Mentions: Because the strength with which bacterial promoters are regulated is a function of the concentration of the relevant transcription factors (Buchler et al, 2003; Bintu et al, 2005a, 2005b), we next attempt to detect the local enhancement of LacI concentration near the lacI gene through the repression strength of a LacI-regulated target. We constructed three families of 14 strains where we inserted into previously constructed lacI-venus strains a cassette bearing a LacI-regulated lacZ target at regular intervals around the genome (Supplementary Figure 1). In Figure 6A–C, the strength of repression is shown as a function of intergenic distance for each family of strains. Colors indicate the growth rate of the culture: slow (red), medium (yellow); fast (green); slow +2 mM IPTG control (black). In each plot, the x axis is adjusted so that the location of the repressor gene lies at the center of the x axis (black vertical dashed lines). In all cases, the strength of repression increases as the target is moved toward the terminus and decreases as the target is moved toward the origin. In each case there is a small but reproducible 1.2–2 × spike in repression when the target is located immediately proximal (∼200 bp downstream) to the repressor gene. We encounter little interference from position-specific effects, with a few exceptions: in all cases, the target inserted near the terminus at the essQ locus (see Supplementary Figure 1) remained unregulated. Additionally, when lacI-venus is inserted near the origin (Figure 6A) the repression strengths at essQ and nth near the terminus are significantly weaker than might otherwise be expected. This may be due to their location within the less densely packed chromosomal crossing region (Wiggins et al, 2010). These data were excluded from the best fit lines in Figure 6A–C.

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