DmK-WT (Milic et al., 2014 ) and hindering load data sets for DmK-3AA (Andreasson et al., 2015 ) are reproduced from our previous work.
The effects of NusA are equivalent to exerting a hindering load of 19 ± 6 pN, which corresponds to an average energy barrier to forward translocation of 2.0 ± 0.4 kB T, a magnitude equivalent to that of the intrinsic translocation barrier of Pol II.
The termination efficiency for his and tR2 terminators did not change with assisting or hindering loads applied on the DNA.
Unloaded velocities of CL constructs with WT-length NL domains were similar, but CL constructs were systematically slower than WT under hindering loads.
Unbinding rates under assisting and moderate hindering loads, ranging from −6 to +20 pN, were obtained directly from velocity and run length records.
Comparison shows that this single mutation can account for some, but not all, of the change in force dependence under hindering loads.
Under hindering loads, both HsK-CL and DmK-CL were appreciably slower, and also more sensitive to load, than either DmK-WT or the corresponding truncated human construct, HsK-WT.
The unloaded release rate (k off) and the associated distance parameter (δoff) were obtained from exponential fits to unbinding data acquired under hindering loads, −25 to 0 pN, and assisting loads, +2 to +20 pN.
The application of hindering loads to the kinesin stalk places a rearward load on its front head, and therefore can serve as a proxy for estimating the rate of detachment of the front head at the unbinding gate.
Unbinding rates under large hindering loads, ranging from −25 to −7 pN, were estimated separately, based on the average dwell time of the last step prior to dissociation (see below).
The velocity decrease under hindering loads is associated with this transition, which we model as k1, with a correspondingly large distance parameter (δ1 ≥ 4 nm; Table 2), equivalent to nearly half the size of the kinesin step.
