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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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Synthesis of DNA construct.A) Schematic of the procedure for construction of the plasmid with two Lac repressor binding sites. (B) Schematic of the protocol for producing labeled DNA using a PCR reaction with labeled primers.
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pone-0005621-g013: Synthesis of DNA construct.A) Schematic of the procedure for construction of the plasmid with two Lac repressor binding sites. (B) Schematic of the protocol for producing labeled DNA using a PCR reaction with labeled primers.

Mentions: Plasmid DNAs, bearing two Lac repressor binding sites spaced at a designed distance, are created using a point mutation method (QuikChange site-directed mutagenesis, Stratagene) on plasmid pUC19. Plasmid pUC19 was chosen as a starting template because it is not only a high copy plasmid but also contains two Lac repressor binding sites: and . The procedure for creating two binding sites separated by the desired distance from template pUC19 is illustrated in fig. 13(A). We first mutate six basepairs in the site converting it to in a way that eliminates the binding affinity for this site [86]. The resulting plasmid is called pUC19O1 indicating it only has a single site. To construct another binding site on the pUC19O1 plasmid, we replace 20 bp with the Lac repressor binding sequence at a series of locations differing by 1 bp increments in their distance from using the mutagenesis method again. For some of the secondary site construction, we have to use either deletion or addition from already made plasmids with two designed binding sites. The details on primers and templates used in this process are listed in Table 2. The final product contains two binding sites and spaced at the desired distance.


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)

Synthesis of DNA construct.A) Schematic of the procedure for construction of the plasmid with two Lac repressor binding sites. (B) Schematic of the protocol for producing labeled DNA using a PCR reaction with labeled primers.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005621-g013: Synthesis of DNA construct.A) Schematic of the procedure for construction of the plasmid with two Lac repressor binding sites. (B) Schematic of the protocol for producing labeled DNA using a PCR reaction with labeled primers.
Mentions: Plasmid DNAs, bearing two Lac repressor binding sites spaced at a designed distance, are created using a point mutation method (QuikChange site-directed mutagenesis, Stratagene) on plasmid pUC19. Plasmid pUC19 was chosen as a starting template because it is not only a high copy plasmid but also contains two Lac repressor binding sites: and . The procedure for creating two binding sites separated by the desired distance from template pUC19 is illustrated in fig. 13(A). We first mutate six basepairs in the site converting it to in a way that eliminates the binding affinity for this site [86]. The resulting plasmid is called pUC19O1 indicating it only has a single site. To construct another binding site on the pUC19O1 plasmid, we replace 20 bp with the Lac repressor binding sequence at a series of locations differing by 1 bp increments in their distance from using the mutagenesis method again. For some of the secondary site construction, we have to use either deletion or addition from already made plasmids with two designed binding sites. The details on primers and templates used in this process are listed in Table 2. The final product contains two binding sites and spaced at the desired distance.

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