Figure 4. High-resolution analysis of gyrase dynamics at 1 mM [ATP].

(a) “Bottom-constrained” experimental geometry. During gyrase activity, the angle and height of the bead reflect changes in Lk and extension of the lower DNA segment^3,6. Measurements were performed using 140 nm rotors under 1.1 pN of tension, and angle and z were both lowpass filtered to 50 Hz. (b) Excised traces of single-enzyme bursts. Angle is plotted as a function of time, with color (blue to red) indicating the instantaneous value of DNA extension. Dominant dwells are visible every two rotations as expected⁶. Short regions flanking each burst show the extension of free DNA. Asterisks mark events in which the DNA extension briefly increases, primarily coinciding with entrance into a new rotational dwell. (c) A single gyrase trace is shown projected along angle and z-axes; brief excursions (*) are visible between dominant dwells. Color represents time, progressing from blue to orange. (d) 2D histogram of angle and z coordinates⁶ accumulated over a total of 97 gyrase cycles belonging to 19 enzymatic bursts on five separate DNA tethers; 1.5 s of data flanking each burst were included to show the position of free DNA. (e) Proposed model explaining excursions in extension (*). DNA contour length is released from one or both CTDs after strand passage, entering a newly defined ν state. The DNA is then recaptured to reset the enzyme for the next cycle.