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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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States and weights for the Lac repressor-DNA system [42].Each of the five state classes shown in the left column has a corresponding statistical weight given by the product of the Boltzmann factor and the microscopic degeneracy of the state. All of the weights have been normalized by the weight of the state in which the DNA is unoccupied. State (v) is treated as a single looped state, even though there are multiple distinct looped configurations. The third column shows how to write these statistical weights in the language of equilibrium constants and . The derivation of these weights and the relation between the statistical mechanical and thermodynamic perspectives can be found in the Supporting Information S1.
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pone-0005621-g002: States and weights for the Lac repressor-DNA system [42].Each of the five state classes shown in the left column has a corresponding statistical weight given by the product of the Boltzmann factor and the microscopic degeneracy of the state. All of the weights have been normalized by the weight of the state in which the DNA is unoccupied. State (v) is treated as a single looped state, even though there are multiple distinct looped configurations. The third column shows how to write these statistical weights in the language of equilibrium constants and . The derivation of these weights and the relation between the statistical mechanical and thermodynamic perspectives can be found in the Supporting Information S1.

Mentions: In order to extract quantities such as the free energy of looping associated with repressor binding (or equivalently, a for looping, essentially the concentration at which in a solution of DNA with sticky ends, the probability of forming circles and dimers is equal) and to examine how the propensity for looping depends upon the number of repressors, we needed looping data at a number of different concentrations. At very low concentration, we expect that there will be negligible looping because neither of the operators will be bound by Lac repressor. At intermediate concentrations, the equilibrium will be dominated by states in which a single repressor tetramer is bound to the DNA at the strong operator, punctuated by transient looping events. In the very high concentration limit, each operator will be occupied by a tetramer (see fig. 2 below), making the formation of a loop nearly impossible.


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)

States and weights for the Lac repressor-DNA system [42].Each of the five state classes shown in the left column has a corresponding statistical weight given by the product of the Boltzmann factor and the microscopic degeneracy of the state. All of the weights have been normalized by the weight of the state in which the DNA is unoccupied. State (v) is treated as a single looped state, even though there are multiple distinct looped configurations. The third column shows how to write these statistical weights in the language of equilibrium constants and . The derivation of these weights and the relation between the statistical mechanical and thermodynamic perspectives can be found in the Supporting Information S1.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005621-g002: States and weights for the Lac repressor-DNA system [42].Each of the five state classes shown in the left column has a corresponding statistical weight given by the product of the Boltzmann factor and the microscopic degeneracy of the state. All of the weights have been normalized by the weight of the state in which the DNA is unoccupied. State (v) is treated as a single looped state, even though there are multiple distinct looped configurations. The third column shows how to write these statistical weights in the language of equilibrium constants and . The derivation of these weights and the relation between the statistical mechanical and thermodynamic perspectives can be found in the Supporting Information S1.
Mentions: In order to extract quantities such as the free energy of looping associated with repressor binding (or equivalently, a for looping, essentially the concentration at which in a solution of DNA with sticky ends, the probability of forming circles and dimers is equal) and to examine how the propensity for looping depends upon the number of repressors, we needed looping data at a number of different concentrations. At very low concentration, we expect that there will be negligible looping because neither of the operators will be bound by Lac repressor. At intermediate concentrations, the equilibrium will be dominated by states in which a single repressor tetramer is bound to the DNA at the strong operator, punctuated by transient looping events. In the very high concentration limit, each operator will be occupied by a tetramer (see fig. 2 below), making the formation of a loop nearly impossible.

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