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Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion

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 D...

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Detalles Bibliográficos
Autores principales: Vanzi, Francesco, Broggio, Chiara, Sacconi, Leonardo, Pavone, Francesco Saverio
Formato: Texto
Lenguaje:English
Publicado: Oxford University Press 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1524907/
https://www.ncbi.nlm.nih.gov/pubmed/16835309
http://dx.doi.org/10.1093/nar/gkl393
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author Vanzi, Francesco
Broggio, Chiara
Sacconi, Leonardo
Pavone, Francesco Saverio
author_facet Vanzi, Francesco
Broggio, Chiara
Sacconi, Leonardo
Pavone, Francesco Saverio
author_sort Vanzi, Francesco
collection PubMed
description 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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spelling pubmed-15249072006-08-09 Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion Vanzi, Francesco Broggio, Chiara Sacconi, Leonardo Pavone, Francesco Saverio Nucleic Acids Res Article 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. Oxford University Press 2006 2006-07-11 /pmc/articles/PMC1524907/ /pubmed/16835309 http://dx.doi.org/10.1093/nar/gkl393 Text en © 2006 The Author(s)
spellingShingle Article
Vanzi, Francesco
Broggio, Chiara
Sacconi, Leonardo
Pavone, Francesco Saverio
Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
title Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
title_full Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
title_fullStr Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
title_full_unstemmed Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
title_short Lac repressor hinge flexibility and DNA looping: single molecule kinetics by tethered particle motion
title_sort lac repressor hinge flexibility and dna looping: single molecule kinetics by tethered particle motion
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1524907/
https://www.ncbi.nlm.nih.gov/pubmed/16835309
http://dx.doi.org/10.1093/nar/gkl393
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AT sacconileonardo lacrepressorhingeflexibilityanddnaloopingsinglemoleculekineticsbytetheredparticlemotion
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