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Concentration and length dependence of DNA looping in transcriptional regulation.

Han L, Garcia HG, Blumberg S, Towles KB, Beausang JF, Nelson PC, Phillips R - PLoS ONE (2009)

Bottom Line: This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter.We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA.The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physics, California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT
In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

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Theory and experiment for the probability density functions of RMS bead excursion for (A) our six “long chain” constructs and (B) our three “short chain” constructs.Blue dashed curves show the data in fig. 7, represented as sums of three Gaussians. Black curves show our theoretically predicted distributions. Because our simulation results were not fits to the data, they did not reproduce perfectly the ratio of looped to unlooped occupancies. For visualization, therefore, we have adjusted this overall ratio by a factor common to all six curves. This rescaling does not affect the locations of the peaks, the relative weights of the two looped-state peaks, nor the dependences of weights on loop length , all of which are zero-fit-parameter predictions of our model. The model yields these histograms as the sum of five contributions, corresponding to the four looped topologies and the unlooped state. The topologies correspond to the different geometries shown in fig. 5. The separate RMS displacements for each individual loop topology for the 89 bp case in (A) and for the 300 bp case in (B) are also shown, labeled according to the scheme in [36].
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pone-0005621-g011: Theory and experiment for the probability density functions of RMS bead excursion for (A) our six “long chain” constructs and (B) our three “short chain” constructs.Blue dashed curves show the data in fig. 7, represented as sums of three Gaussians. Black curves show our theoretically predicted distributions. Because our simulation results were not fits to the data, they did not reproduce perfectly the ratio of looped to unlooped occupancies. For visualization, therefore, we have adjusted this overall ratio by a factor common to all six curves. This rescaling does not affect the locations of the peaks, the relative weights of the two looped-state peaks, nor the dependences of weights on loop length , all of which are zero-fit-parameter predictions of our model. The model yields these histograms as the sum of five contributions, corresponding to the four looped topologies and the unlooped state. The topologies correspond to the different geometries shown in fig. 5. The separate RMS displacements for each individual loop topology for the 89 bp case in (A) and for the 300 bp case in (B) are also shown, labeled according to the scheme in [36].

Mentions: (A) Schematic of the TPM experiment. (B) Scatter plot of drift-corrected positional data. Each dot corresponds to the instantaneous projected position of the bead at a particular instant in time. (C) Running average of Gaussian filtered RMS motion over an effective window of 4 seconds. is the distance from the bead center (dots in panel (B)) to the tether attachment point (centroid of all dots in panel (B)). Red (solid) and green (dashed) lines represent naively expected motion, based on calibration measurements [91] in the absence of any DNA binding protein, for 901 bp DNA and an imagined DNA for which 305+20.5 bp (the center to center distance between operators) are subtracted off of the full length 901 bp tether. (Fig. 11 gives a more precise prediction of the expected excursions in looped states.) (D) Histogram of the RMS motion. Different peaks correspond to looped (labeled B, bottom, and M, middle) or unlooped (labeled U) states. The lines shown here are the same as those shown in (C). The presence of Lac repressor results in a shift of the excursion of the unlooped state with respect to the excursion expected from the protein-free calibration curve. This is reflected in the fact that the U peak does not coincide with the red line. The DNA used here is pUC305L1 (see Materials and Methods section) with 100 pM Lac repressor. A detailed discussion of how to go from microscopy images of beads to traces and histograms like those shown here is given in the movie S1 in the Supporting Information.


Concentration and length dependence of DNA looping in transcriptional regulation.

Han L, Garcia HG, Blumberg S, Towles KB, Beausang JF, Nelson PC, Phillips R - PLoS ONE (2009)

Theory and experiment for the probability density functions of RMS bead excursion for (A) our six “long chain” constructs and (B) our three “short chain” constructs.Blue dashed curves show the data in fig. 7, represented as sums of three Gaussians. Black curves show our theoretically predicted distributions. Because our simulation results were not fits to the data, they did not reproduce perfectly the ratio of looped to unlooped occupancies. For visualization, therefore, we have adjusted this overall ratio by a factor common to all six curves. This rescaling does not affect the locations of the peaks, the relative weights of the two looped-state peaks, nor the dependences of weights on loop length , all of which are zero-fit-parameter predictions of our model. The model yields these histograms as the sum of five contributions, corresponding to the four looped topologies and the unlooped state. The topologies correspond to the different geometries shown in fig. 5. The separate RMS displacements for each individual loop topology for the 89 bp case in (A) and for the 300 bp case in (B) are also shown, labeled according to the scheme in [36].
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682762&req=5

pone-0005621-g011: Theory and experiment for the probability density functions of RMS bead excursion for (A) our six “long chain” constructs and (B) our three “short chain” constructs.Blue dashed curves show the data in fig. 7, represented as sums of three Gaussians. Black curves show our theoretically predicted distributions. Because our simulation results were not fits to the data, they did not reproduce perfectly the ratio of looped to unlooped occupancies. For visualization, therefore, we have adjusted this overall ratio by a factor common to all six curves. This rescaling does not affect the locations of the peaks, the relative weights of the two looped-state peaks, nor the dependences of weights on loop length , all of which are zero-fit-parameter predictions of our model. The model yields these histograms as the sum of five contributions, corresponding to the four looped topologies and the unlooped state. The topologies correspond to the different geometries shown in fig. 5. The separate RMS displacements for each individual loop topology for the 89 bp case in (A) and for the 300 bp case in (B) are also shown, labeled according to the scheme in [36].
Mentions: (A) Schematic of the TPM experiment. (B) Scatter plot of drift-corrected positional data. Each dot corresponds to the instantaneous projected position of the bead at a particular instant in time. (C) Running average of Gaussian filtered RMS motion over an effective window of 4 seconds. is the distance from the bead center (dots in panel (B)) to the tether attachment point (centroid of all dots in panel (B)). Red (solid) and green (dashed) lines represent naively expected motion, based on calibration measurements [91] in the absence of any DNA binding protein, for 901 bp DNA and an imagined DNA for which 305+20.5 bp (the center to center distance between operators) are subtracted off of the full length 901 bp tether. (Fig. 11 gives a more precise prediction of the expected excursions in looped states.) (D) Histogram of the RMS motion. Different peaks correspond to looped (labeled B, bottom, and M, middle) or unlooped (labeled U) states. The lines shown here are the same as those shown in (C). The presence of Lac repressor results in a shift of the excursion of the unlooped state with respect to the excursion expected from the protein-free calibration curve. This is reflected in the fact that the U peak does not coincide with the red line. The DNA used here is pUC305L1 (see Materials and Methods section) with 100 pM Lac repressor. A detailed discussion of how to go from microscopy images of beads to traces and histograms like those shown here is given in the movie S1 in the Supporting Information.

Bottom Line: This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter.We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA.The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

View Article: PubMed Central - PubMed

Affiliation: Department of Applied Physics, California Institute of Technology, Pasadena, California, United States of America.

ABSTRACT
In many cases, transcriptional regulation involves the binding of transcription factors at sites on the DNA that are not immediately adjacent to the promoter of interest. This action at a distance is often mediated by the formation of DNA loops: Binding at two or more sites on the DNA results in the formation of a loop, which can bring the transcription factor into the immediate neighborhood of the relevant promoter. These processes are important in settings ranging from the historic bacterial examples (bacterial metabolism and the lytic-lysogeny decision in bacteriophage), to the modern concept of gene regulation to regulatory processes central to pattern formation during development of multicellular organisms. Though there have been a variety of insights into the combinatorial aspects of transcriptional control, the mechanism of DNA looping as an agent of combinatorial control in both prokaryotes and eukaryotes remains unclear. We use single-molecule techniques to dissect DNA looping in the lac operon. In particular, we measure the propensity for DNA looping by the Lac repressor as a function of the concentration of repressor protein and as a function of the distance between repressor binding sites. As with earlier single-molecule studies, we find (at least) two distinct looped states and demonstrate that the presence of these two states depends both upon the concentration of repressor protein and the distance between the two repressor binding sites. We find that loops form even at interoperator spacings considerably shorter than the DNA persistence length, without the intervention of any other proteins to prebend the DNA. The concentration measurements also permit us to use a simple statistical mechanical model of DNA loop formation to determine the free energy of DNA looping, or equivalently, the for looping.

Show MeSH
Related in: MedlinePlus