Supercoiling and Denaturation in Gal Repressor/Heat Unstable Nucleoid Protein (HU)-Mediated DNA Looping

The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal op...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2003-09, Vol.100 (20), p.11373-11377
Main Authors: Lia, Giuseppe, Bensimon, David, Croquette, Vincent, Allemand, Jean-Francois, Dunlap, David, Dale E. A. Lewis, Adhya, Sankar, Finzi, Laura
Format: Article
Language:English
Subjects:
Biological Sciences
Biophysics
Deoxyribonucleic acid
Dimers
DNA
DNA, Superhelical - chemistry
DNA, Superhelical - genetics
Experiments
Free energy
Galactose - chemistry
Intermediate uveitis
Loops
Molecules
Nucleic Acid Conformation
Nucleoproteins - chemistry
Operons
Plasmids
Protein Denaturation
Proteins
Repression
Repressor Proteins - chemistry
Repressor Proteins - genetics
RNA
Thermodynamics
Topology
Transcription, Genetic
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Summary:The overall topology of DNA profoundly influences the regulation of transcription and is determined by DNA flexibility as well as the binding of proteins that induce DNA torsion, distortion, and/or looping. Gal repressor (GalR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of ≈40 nm of DNA that encompasses the promoters. Associated evidence of a topological regulatory mechanism of the transcription repression is the requirement for a supercoiled DNA template and the histone-like heat unstable nucleoid protein (HU). By using single-molecule manipulations to generate and finely tune tension in DNA molecules, we directly detected GalR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GalR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our single-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.2034851100