Figure 2.

TPM experiments in Ca²⁺. (A) The RMS motions for a single IF190 tether (inverted FokI sites 190 bp apart) in the presence of 5 nM FokI is plotted as a function of time (black trace). The trace shows clear dynamics between an unlooped (high RMS) and a looped (low RMS) state. To find the RMS value of these two states, a threshold was set to divide the data into the two states (step function shown in red) and the dwell times in each state extracted (marked by black arrows). (B) A plot of the cumulative distribution function of the dwell times extracted from the trace in (A) (i.e. the probability of remaining in the looped or the unlooped state as a function of time). The red circles show the dwell times of the looped state and are fitted with a single exponent to obtain the loop release rate (the lifetime of the looped state is unaffected by the protein dissociation step since this has no impact on the RMS values). Since dwell times in the unlooped state are a combination of the protein associating with the recognition sites and the actual loop capture process, the dwell times show a bi-exponential behaviour and are fitted accordingly. The protein dissociation rate can be obtained, despite the fact that it does not give a signal in the RMS trace, by calculating the intersection of the two exponentials in the bi-exponential fit.