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Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping.

Normanno D, Vanzi F, Pavone FS - Nucleic Acids Res. (2008)

Bottom Line: A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed.The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon.In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.

View Article: PubMed Central - PubMed

Affiliation: LENS, European Laboratory for Non-linear Spectroscopy, Università degli Studi di Firenze, Via N. Carrara 1, I-50019 Sesto Fiorentino (FI), Italy. dnormanno@ibec.pcb.ub.es

ABSTRACT
Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between -0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI-DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.

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DNA under torsional constraints. (a) Raw data of a typical measurement of the tether length 〈 R(t) 〉 at different degrees of supercoiling σ. The temporal series of the manipulation is: (1) wrench off, (2) wrench on (with B = 48Gauss), (3) under-winding rotations, in steps of one turn, (4) wrench off, (5) wrench on, (6) over-winding rotations, in steps of one turn, (7) wrench off. (b) Tether length 〈 R 〉 vs. σ. 〈 R 〉 has been obtained by averaging the trace in (a) over all the data acquired for each σ value. 〈 R 〉 decreases linearly for values of /σ/ up to ∼0.026, consistently with the formation of one plectoneme per rotation imposed, as expected in the DNA entropic regime (29). The angular coefficient of the linear fits to the data (red curves) are (−32 ± 1) nm/turn for positive values of σ and (33 ± 2) nm/turn for negative values of σ. The reduction due to the stretching force can be estimated from the data by considering the filled square (indicating the value 〈 R 〉 Boff in the absence of magnetic field) and the value corresponding to σ = 0, which is the tether length when the magnetic wrench is on (B = 48 Gauss). In this case 〈 R〉 σ = 0/ 〈 R 〉 Boff = 0.92, thus the magnetic field induces a reduction of 〈 R 〉 of the order of 10%. The upper scale in the graph reports the number of rotations r added or removed to the DNA molecule; the relationship between r and σ is: σ = r·h/LC, where h = 10.4 bp is the DNA helical repeat in vitro, and LC = 1200 bp is the contour length of the DNA (see ‘Materials and Methods’ section). The right scale of the graph shows the relative extension of the tether length, calculated as 〈 R 〉 / 〈 R 〉 Boff.
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Figure 3: DNA under torsional constraints. (a) Raw data of a typical measurement of the tether length 〈 R(t) 〉 at different degrees of supercoiling σ. The temporal series of the manipulation is: (1) wrench off, (2) wrench on (with B = 48Gauss), (3) under-winding rotations, in steps of one turn, (4) wrench off, (5) wrench on, (6) over-winding rotations, in steps of one turn, (7) wrench off. (b) Tether length 〈 R 〉 vs. σ. 〈 R 〉 has been obtained by averaging the trace in (a) over all the data acquired for each σ value. 〈 R 〉 decreases linearly for values of /σ/ up to ∼0.026, consistently with the formation of one plectoneme per rotation imposed, as expected in the DNA entropic regime (29). The angular coefficient of the linear fits to the data (red curves) are (−32 ± 1) nm/turn for positive values of σ and (33 ± 2) nm/turn for negative values of σ. The reduction due to the stretching force can be estimated from the data by considering the filled square (indicating the value 〈 R 〉 Boff in the absence of magnetic field) and the value corresponding to σ = 0, which is the tether length when the magnetic wrench is on (B = 48 Gauss). In this case 〈 R〉 σ = 0/ 〈 R 〉 Boff = 0.92, thus the magnetic field induces a reduction of 〈 R 〉 of the order of 10%. The upper scale in the graph reports the number of rotations r added or removed to the DNA molecule; the relationship between r and σ is: σ = r·h/LC, where h = 10.4 bp is the DNA helical repeat in vitro, and LC = 1200 bp is the contour length of the DNA (see ‘Materials and Methods’ section). The right scale of the graph shows the relative extension of the tether length, calculated as 〈 R 〉 / 〈 R 〉 Boff.

Mentions: The response of the DNA to externally imposed supercoiling in the combined TPM-magnetic manipulation experiment is reported in Figure 3. Figure 3a shows a recording of 〈 R(t) 〉 during a variety of manipulations (see figure legend for details). The data show the full reversibility of the topological variations induced in the DNA molecule with the magnetic wrench. Figure 3b shows 〈 R 〉 versus σ. In the range of σ explored in the measurements (−0.026 < σ < 0.026), DNA forms plectonemes (one plectoneme is formed or destroyed for each turn added or removed to the DNA molecule) and its extension decreases linearly for increasing /σ/ (as shown by the linear fits, red curves in Figure 3b).Figure 3.


Single-molecule manipulation reveals supercoiling-dependent modulation of lac repressor-mediated DNA looping.

Normanno D, Vanzi F, Pavone FS - Nucleic Acids Res. (2008)

DNA under torsional constraints. (a) Raw data of a typical measurement of the tether length 〈 R(t) 〉 at different degrees of supercoiling σ. The temporal series of the manipulation is: (1) wrench off, (2) wrench on (with B = 48Gauss), (3) under-winding rotations, in steps of one turn, (4) wrench off, (5) wrench on, (6) over-winding rotations, in steps of one turn, (7) wrench off. (b) Tether length 〈 R 〉 vs. σ. 〈 R 〉 has been obtained by averaging the trace in (a) over all the data acquired for each σ value. 〈 R 〉 decreases linearly for values of /σ/ up to ∼0.026, consistently with the formation of one plectoneme per rotation imposed, as expected in the DNA entropic regime (29). The angular coefficient of the linear fits to the data (red curves) are (−32 ± 1) nm/turn for positive values of σ and (33 ± 2) nm/turn for negative values of σ. The reduction due to the stretching force can be estimated from the data by considering the filled square (indicating the value 〈 R 〉 Boff in the absence of magnetic field) and the value corresponding to σ = 0, which is the tether length when the magnetic wrench is on (B = 48 Gauss). In this case 〈 R〉 σ = 0/ 〈 R 〉 Boff = 0.92, thus the magnetic field induces a reduction of 〈 R 〉 of the order of 10%. The upper scale in the graph reports the number of rotations r added or removed to the DNA molecule; the relationship between r and σ is: σ = r·h/LC, where h = 10.4 bp is the DNA helical repeat in vitro, and LC = 1200 bp is the contour length of the DNA (see ‘Materials and Methods’ section). The right scale of the graph shows the relative extension of the tether length, calculated as 〈 R 〉 / 〈 R 〉 Boff.
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Related In: Results  -  Collection

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Figure 3: DNA under torsional constraints. (a) Raw data of a typical measurement of the tether length 〈 R(t) 〉 at different degrees of supercoiling σ. The temporal series of the manipulation is: (1) wrench off, (2) wrench on (with B = 48Gauss), (3) under-winding rotations, in steps of one turn, (4) wrench off, (5) wrench on, (6) over-winding rotations, in steps of one turn, (7) wrench off. (b) Tether length 〈 R 〉 vs. σ. 〈 R 〉 has been obtained by averaging the trace in (a) over all the data acquired for each σ value. 〈 R 〉 decreases linearly for values of /σ/ up to ∼0.026, consistently with the formation of one plectoneme per rotation imposed, as expected in the DNA entropic regime (29). The angular coefficient of the linear fits to the data (red curves) are (−32 ± 1) nm/turn for positive values of σ and (33 ± 2) nm/turn for negative values of σ. The reduction due to the stretching force can be estimated from the data by considering the filled square (indicating the value 〈 R 〉 Boff in the absence of magnetic field) and the value corresponding to σ = 0, which is the tether length when the magnetic wrench is on (B = 48 Gauss). In this case 〈 R〉 σ = 0/ 〈 R 〉 Boff = 0.92, thus the magnetic field induces a reduction of 〈 R 〉 of the order of 10%. The upper scale in the graph reports the number of rotations r added or removed to the DNA molecule; the relationship between r and σ is: σ = r·h/LC, where h = 10.4 bp is the DNA helical repeat in vitro, and LC = 1200 bp is the contour length of the DNA (see ‘Materials and Methods’ section). The right scale of the graph shows the relative extension of the tether length, calculated as 〈 R 〉 / 〈 R 〉 Boff.
Mentions: The response of the DNA to externally imposed supercoiling in the combined TPM-magnetic manipulation experiment is reported in Figure 3. Figure 3a shows a recording of 〈 R(t) 〉 during a variety of manipulations (see figure legend for details). The data show the full reversibility of the topological variations induced in the DNA molecule with the magnetic wrench. Figure 3b shows 〈 R 〉 versus σ. In the range of σ explored in the measurements (−0.026 < σ < 0.026), DNA forms plectonemes (one plectoneme is formed or destroyed for each turn added or removed to the DNA molecule) and its extension decreases linearly for increasing /σ/ (as shown by the linear fits, red curves in Figure 3b).Figure 3.

Bottom Line: A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed.The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon.In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.

View Article: PubMed Central - PubMed

Affiliation: LENS, European Laboratory for Non-linear Spectroscopy, Università degli Studi di Firenze, Via N. Carrara 1, I-50019 Sesto Fiorentino (FI), Italy. dnormanno@ibec.pcb.ub.es

ABSTRACT
Gene expression regulation is a fundamental biological process which deploys specific sets of genomic information depending on physiological or environmental conditions. Several transcription factors (including lac repressor, LacI) are present in the cell at very low copy number and increase their local concentration by binding to multiple sites on DNA and looping the intervening sequence. In this work, we employ single-molecule manipulation to experimentally address the role of DNA supercoiling in the dynamics and stability of LacI-mediated DNA looping. We performed measurements over a range of degrees of supercoiling between -0.026 and +0.026, in the absence of axial stretching forces. A supercoiling-dependent modulation of the lifetimes of both the looped and unlooped states was observed. Our experiments also provide evidence for multiple structural conformations of the LacI-DNA complex, depending on torsional constraints. The supercoiling-dependent modulation demonstrated here adds an important element to the model of the lac operon. In fact, the complex network of proteins acting on the DNA in a living cell constantly modifies its topological and mechanical properties: our observations demonstrate the possibility of establishing a signaling pathway from factors affecting DNA supercoiling to transcription factors responsible for the regulation of specific sets of genes.

Show MeSH
Related in: MedlinePlus