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, and͞or 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 Ϸ40 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 GalR͞HU-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 GalR͞HU-mediated DNA loop formation. Such topological intermediates may operate similarly in other multiprotein complexes of transcription, replication, and recombination. T he nucleoid structure in the bacterium Escherichia coli contains a circular DNA molecule of 4.7 million bp present in highly condensed form. The condensation is mediated by DNA supercoiling and the binding of several small nucleoidassociated proteins, e.g., heat unstable nucleoid protein (HU), integration host factor (IHF), factor for inversion stimulation (FIS), histone-like nucleoid structuring protein (HNS), suppressor of thymidylate synthase mutant phenotype A (StpA), and DNA binding protein from starved cells (Dps). These proteins are known to bend DNA or bind to altered structures of DNA. It is suggested that these proteins are mainly responsible for the compaction of DNA in a way that distinguishes the bacterial nucleoid from eukaryotic chromatin. These proteins are also associated with the machinery of macromolecular biosynthesis, including RNA polymerase and specific gene-regulatory, DNAbinding proteins such as repressors and activators. Indeed, DNA may serve as a scaffold for the organized recruitment and assembly of proteins at specific positions to create nucleoprotein complexes with specific activity and regulatory properties. Such positioning has long been postulated to be the mechanism of repression of the gal operon by the gal repressor dimer protein (GalR). GalR represses transcription initiation from the two promoters, P1 and P2, of the gal operon by binding to two spatially separated operators, O E and O I , which encompass the promoters (1). Repression also requires supercoiled DNA and the presence of the nucleoid-associated protein HU (2). It has been proposed that a DNA loop generated by the interaction of the two operator-bound gal repressors inactivates the promoter (3). Repression of the gal operon would, th...

Recently, it was proposed that DNA looping by the λ repressor (CI protein) strengthens repression of lytic genes during lysogeny and simultaneously ensures efficient switching to lysis. To investigate this hypothesis, tethered particle motion experiments were performed and dynamic CI-mediated looping of single DNA molecules containing the λ repressor binding sites separated by 2317 bp (the wild-type distance) was quantitatively analyzed. DNA containing all three intact operators or with mutated o3 operators were compared. Modeling the thermodynamic data established the free energy of CI octamer-mediated loop formation as 1.7 kcal/mol, which decreased to –0.7 kcal/mol when supplemented by a tetramer (octamer+tetramer-mediated loop). These results support the idea that loops secured by an octamer of CI bound at oL1, oL2, oR1 and oR2 operators must be augmented by a tetramer of CI bound at the oL3 and oR3 to be spontaneous and stable. Thus the o3 sites are critical for loops secured by the CI protein that attenuate cI expression.

SummaryEfficient repression of the two promoters P1 and P 2 of the gal operon requires the formation of a DNA loop encompassing the promoters. In vitro, DNA loopingmediated repression involves binding of the Gal repressor (GalR) to two gal operators (O E and O I ) and binding of the histone-like protein HU to a specific locus (hbs) about the midpoint between O E and O I , and supercoiled DNA. Without DNA looping, GalR binding to O E partially represses P 1 and stimulates P 2. We investigated the requirement for DNA supercoiling and HU in repression of the gal promoters in vivo in strains containing a fusion of a reporter gene, gusA or lacZ, to each promoter individually. While the P 1 promoter was found to be repressible in the absence of DNA supercoiling and HU, the repression of P 2 was entirely dependent upon DNA supercoiling in vivo. The P 2 promoter was fully derepressed when supercoiling was inhibited by the addition of coumermycin in cells. P 2, but not P1, was also totally derepressed by the absence of HU or the O I operator. From these results, we propose that the repression of the gal promoters in vivo is mediated by the formation of a higher order DNAmultiprotein complex containing GalR, HU and supercoiled DNA. In the absence of this complex, P 1 but not P 2 is still repressed by GalR binding to O E . The specific nucleoprotein complexes involving histone-like proteins, which repress promoter activity while remaining sensitive to inducing signals, as discussed, may occur more generally in bacterial nucleoids.