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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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Looping probability , as a function of interoperator spacing.(A) Looping probability for short constructs. (B) Looping probability for one full helical repeat. These probabilities are obtained by averaging over the  of each bead. The error bars correspond to the standard error associated with this magnitude. For more information see Supporting Information.
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pone-0005621-g008: Looping probability , as a function of interoperator spacing.(A) Looping probability for short constructs. (B) Looping probability for one full helical repeat. These probabilities are obtained by averaging over the of each bead. The error bars correspond to the standard error associated with this magnitude. For more information see Supporting Information.

Mentions: The data can be converted into a plot of the dependence of the looping probability on interoperator spacing as shown in fig. 8. This figure shows as a function of the DNA length between the two operators. The looping probability shows a weak dependence on the interoperator spacing but reveals no conclusive signature of phasing; to really detect such phasing with confidence, however, would require more measurements in single basepair increments. The maximum looping is achieved when the two binding sites are 306 bp apart, suggesting that at this distance, the two sites are in an optimal phasing orientation for binding of the two heads of Lac repressor. The ability to form stable out-of-phase (two binding sites are on the opposite side of the DNA) loops with only a small reduction in stability is consistent with previous studies [26]. The relatively stable looping over the entire helical repeat is also consistent with the relatively constant repression level in vivo for similar interoperator spacing [11].


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)

Looping probability , as a function of interoperator spacing.(A) Looping probability for short constructs. (B) Looping probability for one full helical repeat. These probabilities are obtained by averaging over the  of each bead. The error bars correspond to the standard error associated with this magnitude. For more information see Supporting Information.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005621-g008: Looping probability , as a function of interoperator spacing.(A) Looping probability for short constructs. (B) Looping probability for one full helical repeat. These probabilities are obtained by averaging over the of each bead. The error bars correspond to the standard error associated with this magnitude. For more information see Supporting Information.
Mentions: The data can be converted into a plot of the dependence of the looping probability on interoperator spacing as shown in fig. 8. This figure shows as a function of the DNA length between the two operators. The looping probability shows a weak dependence on the interoperator spacing but reveals no conclusive signature of phasing; to really detect such phasing with confidence, however, would require more measurements in single basepair increments. The maximum looping is achieved when the two binding sites are 306 bp apart, suggesting that at this distance, the two sites are in an optimal phasing orientation for binding of the two heads of Lac repressor. The ability to form stable out-of-phase (two binding sites are on the opposite side of the DNA) loops with only a small reduction in stability is consistent with previous studies [26]. The relatively stable looping over the entire helical repeat is also consistent with the relatively constant repression level in vivo for similar interoperator spacing [11].

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