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Nucleic Acids Research Advance Access published online on February 29, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn071
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

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

Davide Normanno1,*, Francesco Vanzi1,2 and Francesco Saverio Pavone1,3

1LENS, European Laboratory for Non-linear Spectroscopy, Università degli Studi di Firenze, Via N. Carrara 1, I-50019 Sesto Fiorentino (FI), 2Dipartimento di Biologia Animale e Genetica ‘Leo Pardi’, Università degli Studi di Firenze, Via Romana 17, I-50125 Firenze (FI) and 3Dipartimento di Fisica, Università degli Studi di Firenze, Via G. Sansone 1, I-50019 Sesto Fiorentino (FI), Italy

*To whom correspondence should be addressed. Tel: +34 669 231 321; Fax: +34 93 402 0183; Email: dnormanno{at}ibec.pcb.ub.es

Received November 6, 2007. Revised January 7, 2008. Accepted February 5, 2008.

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.

Present address: Davide Normanno, IBEC - Institut de Bioenginyeria de Catalunya, Parc Científic de Barcelona, Baldiri Reixac, 15-21 E-08028 Barcelona, Spain


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