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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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Length dependence of DNA looping.(A) Histogram of the tethered Brownian motion for DNAs with two Lac repressor binding sites spaced from  (bottom) to 310 bp (top). (B) Histogram of the Brownian motion for DNAs with two Lac repressor binding sites spaced at , 94 and 100 bp. The two dashed lines represent the naively expected motion based on our calibration measurements for the full length tether and the same DNA when the center to center distance between operators is subtracted from the tether length. (Again see also fig. 11.) Representative traces for each of the lengths shown here can be found in the Supporting Information.
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pone-0005621-g007: Length dependence of DNA looping.(A) Histogram of the tethered Brownian motion for DNAs with two Lac repressor binding sites spaced from (bottom) to 310 bp (top). (B) Histogram of the Brownian motion for DNAs with two Lac repressor binding sites spaced at , 94 and 100 bp. The two dashed lines represent the naively expected motion based on our calibration measurements for the full length tether and the same DNA when the center to center distance between operators is subtracted from the tether length. (Again see also fig. 11.) Representative traces for each of the lengths shown here can be found in the Supporting Information.

Mentions: The beautiful in vivo repression experiments of [11] demonstrate that the length of the DNA loop formed by Lac repressor strongly affects the probability of loop formation (especially for loop lengths less than 150 bp). In particular, those authors (and others) [12], [13], [64], [65] have observed “phasing”: The relative orientations of the two operators changes the ease with which repressor can loop. Similar phasing effects have been observed in in vitro cyclization assays [3], [34], [66], [67]. What has not been clear is how to concretely and quantitatively relate these results on DNA mechanics from the in vivo and in vitro settings. Our idea was to systematically examine the same progression of DNA lengths that have been observed in vivo, but now using TPM experiments. To that end, we have measured TPM trajectories for a series of interoperator spacings measured in 1 bp increments. The results of this systematic series of measurements for DNAs harboring operators spaced over the range are shown in fig. 7 (as are the results for several shorter lengths to be discussed in the next section). Each plot shows the probability of the three states for a particular interoperator spacing.


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)

Length dependence of DNA looping.(A) Histogram of the tethered Brownian motion for DNAs with two Lac repressor binding sites spaced from  (bottom) to 310 bp (top). (B) Histogram of the Brownian motion for DNAs with two Lac repressor binding sites spaced at , 94 and 100 bp. The two dashed lines represent the naively expected motion based on our calibration measurements for the full length tether and the same DNA when the center to center distance between operators is subtracted from the tether length. (Again see also fig. 11.) Representative traces for each of the lengths shown here can be found in the Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2682762&req=5

pone-0005621-g007: Length dependence of DNA looping.(A) Histogram of the tethered Brownian motion for DNAs with two Lac repressor binding sites spaced from (bottom) to 310 bp (top). (B) Histogram of the Brownian motion for DNAs with two Lac repressor binding sites spaced at , 94 and 100 bp. The two dashed lines represent the naively expected motion based on our calibration measurements for the full length tether and the same DNA when the center to center distance between operators is subtracted from the tether length. (Again see also fig. 11.) Representative traces for each of the lengths shown here can be found in the Supporting Information.
Mentions: The beautiful in vivo repression experiments of [11] demonstrate that the length of the DNA loop formed by Lac repressor strongly affects the probability of loop formation (especially for loop lengths less than 150 bp). In particular, those authors (and others) [12], [13], [64], [65] have observed “phasing”: The relative orientations of the two operators changes the ease with which repressor can loop. Similar phasing effects have been observed in in vitro cyclization assays [3], [34], [66], [67]. What has not been clear is how to concretely and quantitatively relate these results on DNA mechanics from the in vivo and in vitro settings. Our idea was to systematically examine the same progression of DNA lengths that have been observed in vivo, but now using TPM experiments. To that end, we have measured TPM trajectories for a series of interoperator spacings measured in 1 bp increments. The results of this systematic series of measurements for DNAs harboring operators spaced over the range are shown in fig. 7 (as are the results for several shorter lengths to be discussed in the next section). Each plot shows the probability of the three states for a particular interoperator spacing.

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