Probing DNA Topology Using Tethered Particle Motion

Transcription factors mediate the formation of nucleoprotein complexes that are critical for efficient regulation of epigenetic switches. In these complexes, DNA is frequently bent or looped by the protein; other times, strong interactions lead the DNA to fully wrap the regulatory protein(s). The equilibrium between the bending, looping, full and partial wrapping of DNA governs the level of transcriptional regulation and is tuned by biophysical parameters. Characterization of the structure, kinetics, and thermodynamics of formation of such nucleoprotein complexes is fundamental to the understanding of the molecular mechanisms that underlie the operation of the genetic switches controlled by them. Here, we describe in detail how to perform tethered particle motion experiments aimed at understanding how protein–DNA interactions influence the formation and breakdown of these regulatory complexes.