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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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Concentration dependence of the distribution of bead excursions.The histograms show the distribution of RMS motions averaged over 4 seconds at different concentrations of Lac repressor. The blue histograms correspond to measurements for a length between operators of  (see fig. 15), whereas the red histogram is a control where  has been deleted. The two dashed lines represent the naively expected motion, based on our calibration measurements [91]. (See fig. 11 for a more precise prediction of the peak locations.)
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pone-0005621-g003: Concentration dependence of the distribution of bead excursions.The histograms show the distribution of RMS motions averaged over 4 seconds at different concentrations of Lac repressor. The blue histograms correspond to measurements for a length between operators of (see fig. 15), whereas the red histogram is a control where has been deleted. The two dashed lines represent the naively expected motion, based on our calibration measurements [91]. (See fig. 11 for a more precise prediction of the peak locations.)

Mentions: This progression of qualitative behavior is indeed seen in fig. 3, which shows data from eight distinct concentrations of Lac repressor, as well as a single-operator control in which the DNA lacks a secondary operator. Throughout this work we define sequence length or loop length as the end-to-end distance between the operators as shown in fig. 15. These curves correspond to a sequence length of 306 bp and are generated by summing the normalized histograms from all of the individual trajectories for each concentration that pass our bead selection criteria (bead selection criteria are discussed in detail in the Supporting Information S1). A key feature of these data is the way in which the two looped states are turned off as the concentration of Lac repressor is increased to very high levels. This phenomenon is expected since the Lac repressor exists always as tetramers under the conditions used here [47], [48], and competition for binding at the second operator between loose Lac repressor and Lac repressor bound to the other operator is stronger as the concentration of Lac repressor increases. However, the two different looped species have slightly different responses at high repressor concentrations. For example, at 1 nM concentrations, the intermediate looped state has become very infrequent, whereas the shortest looped state remains competitive. Similar concentration dependence of Lac repressor mediated DNA looping was studied previously [24] at 4 pM, 20 pM and 100 pM. Those experiments revealed that looping is suppressed as the concentration goes up.


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)

Concentration dependence of the distribution of bead excursions.The histograms show the distribution of RMS motions averaged over 4 seconds at different concentrations of Lac repressor. The blue histograms correspond to measurements for a length between operators of  (see fig. 15), whereas the red histogram is a control where  has been deleted. The two dashed lines represent the naively expected motion, based on our calibration measurements [91]. (See fig. 11 for a more precise prediction of the peak locations.)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2682762&req=5

pone-0005621-g003: Concentration dependence of the distribution of bead excursions.The histograms show the distribution of RMS motions averaged over 4 seconds at different concentrations of Lac repressor. The blue histograms correspond to measurements for a length between operators of (see fig. 15), whereas the red histogram is a control where has been deleted. The two dashed lines represent the naively expected motion, based on our calibration measurements [91]. (See fig. 11 for a more precise prediction of the peak locations.)
Mentions: This progression of qualitative behavior is indeed seen in fig. 3, which shows data from eight distinct concentrations of Lac repressor, as well as a single-operator control in which the DNA lacks a secondary operator. Throughout this work we define sequence length or loop length as the end-to-end distance between the operators as shown in fig. 15. These curves correspond to a sequence length of 306 bp and are generated by summing the normalized histograms from all of the individual trajectories for each concentration that pass our bead selection criteria (bead selection criteria are discussed in detail in the Supporting Information S1). A key feature of these data is the way in which the two looped states are turned off as the concentration of Lac repressor is increased to very high levels. This phenomenon is expected since the Lac repressor exists always as tetramers under the conditions used here [47], [48], and competition for binding at the second operator between loose Lac repressor and Lac repressor bound to the other operator is stronger as the concentration of Lac repressor increases. However, the two different looped species have slightly different responses at high repressor concentrations. For example, at 1 nM concentrations, the intermediate looped state has become very infrequent, whereas the shortest looped state remains competitive. Similar concentration dependence of Lac repressor mediated DNA looping was studied previously [24] at 4 pM, 20 pM and 100 pM. Those experiments revealed that looping is suppressed as the concentration goes up.

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