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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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Promoter regions of the different short loop constructs.(A) Promoter region of pZS25-YFP which has a variant of the lacUV5 promoter and an  operator upstream overlapping the −35 region. (B) Final construct that allows to insert arbitrary DNA sequences between a  and  operators.
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pone-0005621-g014: Promoter regions of the different short loop constructs.(A) Promoter region of pZS25-YFP which has a variant of the lacUV5 promoter and an operator upstream overlapping the −35 region. (B) Final construct that allows to insert arbitrary DNA sequences between a and operators.

Mentions: A variant of the lacUV5 promoter [10] was synthesized and placed between the EcoRI and XhoI sites of pZS22-YFP in order to create pZS25'-YFP. This promoter included the −35 and −10 regions of the lacUV5 promoter, an AseI site between the two signals and a operator at position −45 from the transcription start as shown in fig. 14(A).


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)

Promoter regions of the different short loop constructs.(A) Promoter region of pZS25-YFP which has a variant of the lacUV5 promoter and an  operator upstream overlapping the −35 region. (B) Final construct that allows to insert arbitrary DNA sequences between a  and  operators.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005621-g014: Promoter regions of the different short loop constructs.(A) Promoter region of pZS25-YFP which has a variant of the lacUV5 promoter and an operator upstream overlapping the −35 region. (B) Final construct that allows to insert arbitrary DNA sequences between a and operators.
Mentions: A variant of the lacUV5 promoter [10] was synthesized and placed between the EcoRI and XhoI sites of pZS22-YFP in order to create pZS25'-YFP. This promoter included the −35 and −10 regions of the lacUV5 promoter, an AseI site between the two signals and a operator at position −45 from the transcription start as shown in fig. 14(A).

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