FIGURE 3 Single-molecule analysis determines the velocity of an RNA polymerase along a DNA and monitors pausing. (a) Representation of the experimental setup (not to scale). Transcribing RNA polymerase with nascent RNA is chemically attached to a polystyrene bead, and the upstream end of the duplex DNA is attached through an antibody linkage to a moveable microscope stage. The bead is held by a laser trap at a predetermined position, which results in a restoring force exerted on the bead. (b) Representative record of position and velocity for a single polymerase molecule transcribing a 3,500 bp DNA template with <18 piconewtons of hindering force. Pausing occurs on multiple timescales, with distinct pauses of seconds-long duration and shorter pauses of about 1 second, as seen in the expanded portion of the trace (arrows point to times when the polymerase transitions from paused to elongating state).