Published online 11 July 2006
© 2006 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-commerical use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
1 LENS—European Laboratory for Nonlinear Spectroscopy, University of Florence Italy 2 Department of Animal Biology and Genetics ‘Leo Pardi’, University of Florence Italy 3 Department of Physics, University of Trento Italy 4 Department of Physics, University of Florence Italy
*To whom correspondence should be addressed at LENS—Via Nello Carrara 1, 50019 Sesto Fiorentino (FI)—Italy. Tel: +39 055 457 2476; Fax: +39 055 457 2451; Email: fvanzi{at}lens.unifi.it
Received February 15, 2006. Revised April 13, 2006. Accepted May 9, 2006.
The tethered particle motion (TPM) allows the direct detection of activity of a variety of biomolecules at the single molecule level. First pioneered for RNA polymerase, it has recently been applied also to other enzymes. In this work we employ TPM for a systematic investigation of the kinetics of DNA looping by wild-type Lac repressor (wt-LacI) and by hinge mutants Q60G and Q60 + 1. We implement a novel method for TPM data analysis to reliably measure the kinetics of loop formation and disruption and to quantify the effects of the protein hinge flexibility and of DNA loop strain on such kinetics. We demonstrate that the flexibility of the protein hinge has a profound effect on the lifetime of the looped state. Our measurements also show that the DNA bending energy plays a minor role on loop disruption kinetics, while a strong effect is seen on the kinetics of loop formation. These observations substantiate the growing number of theoretical studies aimed at characterizing the effects of DNA flexibility, tension and torsion on the kinetics of protein binding and dissociation, strengthening the idea that these mechanical factors in vivo may play an important role in the modulation of gene expression regulation.
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