Recent developments on fluorescent proteins and microscopy techniques have allowed the probing of single molecules in a living bacterial cell with high specificity, millisecond time resolution, and nanometer spatial precision. Recording movies and analyzing dynamics of individual macromolecules have brought new insights into the mechanisms of many processes in molecular biology, such as DNA-protein interactions, gene regulation, transcription, translation, and replication, among others. Here we review the key methods of single-molecule detection and highlight numerous examples to illustrate how these experiments are contributing to the quantitative understanding of the fundamental processes in a living cell.

The Snf2 family represents a functionally diverse class of ATPase sharing the ability to modify DNA structure. Here, we use a magnetic trap and an atomic force microscope to monitor the activity of a member of this class: the RSC complex. This enzyme caused transient shortenings in DNA length involving translocation of typically 400 bp within 2 s, resulting in the formation of a loop whose size depended on both the force applied to the DNA and the ATP concentration. The majority of loops then decrease in size within a time similar to that with which they are formed, suggesting that the motor has the ability to reverse its direction. Loop formation was also associated with the generation of negative DNA supercoils. These observations support the idea that the ATPase motors of the Snf2 family of proteins act as DNA translocases specialized to generate transient distortions in DNA structure.

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...