Supercoiling and denaturation in Gal repressor/heat unstable nucleoid protein (HU)-mediated DNA looping

Publication Type:

Journal Article

Source:

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, NATL ACAD SCIENCES, Volume 100, Number 20, 2101 CONSTITUTION AVE NW, WASHINGTON, DC 20418 USA, p.11373-11377 (2003)

DOI:

10.1073/pnas.2034851100

Keywords:

AFFINITY; CONTAINS; ESCHERICHIA-COLI; HU PROTEIN; MODEL; SEQUENCE; SITE-SPECIFIC BINDING; SPECIFICITY; STRAND BINDING-PROTEIN; TRANSCRIPTION

Abstract:

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 (GaIR) is thought to repress transcription from the two promoters of the gal operon of Escherichia coli by forming a DNA loop of approximate to40 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 GaIR/HU-mediated DNA looping and characterized its kinetics, thermodynamics, and supercoiling dependence. The factors required for gal DNA looping in single-molecule experiments (HU, GaIR and DNA supercoiling) correspond exactly to those necessary for gal repression observed both in vitro and in vivo. Our sing e-molecule experiments revealed that negatively supercoiled DNA, under slight tension, denatured to facilitate GaIR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination.