Book of Abstracts: Albany 2003
June 17-21 2003
Single Molecule Supercoiling and Denaturation in GalR/HU-mediated DNA Looping
Gene regulation is tightly controlled and loss of this control may lead to aberrant cell growth as it is found in cancer. During transcription the interaction of RNA polymerase with the promoter and with regulators is modulated in part by the overall topology of the DNA template. The flexibility of the DNA greatly influences this topology and DNA-binding proteins that supercoil, bend or loop DNA can alter the elastic properties of DNA. Furthermore, the interplay between supercoiling and DNA bending proteins provides a general control of gene expression related to the physiological status of the cells. In order to reach a better understanding of the molecular mechanisms of transcription regulation, we have chosen the gal operon as the simplest and best described system in which looping and supercoiling are intimately related. In the presence of supercoiling, two architectural proteins: GalR and HU act in concert to repress transcription of the galactose operon in Escherichia coli. By delicately stretching and unwinding single DNA molecules, we directly detected GalR/HU-mediated DNA looping. Similarly to in vivo transcription experiments, Gal repressor, the HU protein, and DNA supercoiling were all required for looping. The free energy change measured for looping revealed remarkable stabilization by the HU protein. The SSB protein was shown to interfere with looping, which suggests that negatively supercoiled DNA, under slight tension, denatures to facilitate GalR/HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multi-protein complexes of transcription, replication, and recombination.
Department of Biology