Figure 2.

Single-molecule fluorescence methods. Fluorescence may be used to track binding and residence times of accessory factors (upper panel) or the position of the RNAP holoenzyme by covalently attaching a fluorescent dye (yellow star) and exciting it with an appropriate wavelength (wavy blue arrows). FRET (lower panel) allows the determination of intramolecular distances through fluorescent coupling between a donor (yellow star) and an acceptor (red star) dye. In the lower left panel, the donor (yellow) is excited (blue arrows) and emits light. When the donor fluorophore moves sufficiently close to the acceptor (lower right), resonance energy transfer results in emission of a longer wavelength by the acceptor. The degree of acceptor emission relative to donor excitation is sensitive to the distance between the attached dyes.