(Laser) lights, camera, action: discovering new gene-expression mechanisms by filming single molecules

Single-molecule fluorescence measurements bypass ensemble averaging to offer unprecedented direct access to biologically important heterogeneity and dynamics; this is true both for in vitro reactions using purified biochemical systems, and for reactions inside the true biological milieu of living cells. During the past few years, we have developed and used such methods (namely single-molecule FRET, super-resolution imaging, and single-particle tracking methods) to answer long-standing questions in transcription and to uncover new mechanisms in gene expression and DNA repair. I will discuss examples of our in vitro work focusing on the discovery of pausing during initial transcription, the direct observation of sub-nanometer conformational changes of important dynamic modules of the RNA polymerase machinery and their control by antibiotics, and if time permits, our recent results that establish an intriguing new model for the mechanism of DNA opening in transcription initiation. I will also discuss examples of our in vivo single-molecule work inside living bacteria, including the exploration of the relation between chromosome organization and gene expression in the context of different bacterial physiological states. Our methods and biological observations are general and should apply to many.