Runs under assisting load conditions for DmK-5AA and DmK-6AA were collected using an improved force clamp (Milic et al., 2014).
However, the finding that assisting load fails to appreciably increase the WT velocity suggests that the rate of rear-head release by WT kinesin must be substantially higher than the rate-limiting step(s) of the mechanochemical cycle.
We strongly favor the former possibility, because it also helps to explain the observed decrease in run length under assisting load, as well as the force dependence of exponential fits to the run length data.
(A ) Mean run lengths (mean ± SE; N = 84 210) under moderate assisting load (+4 pN), in the presence of nucleotide analogs (green), 100 mM potassium chloride (KCl; purple), or 100 mM potassium phosphate (KPi; orange).
In contrast to the factors discussed above, optical tweezers experiment demonstrated that E. coli NusA slowed the pause-free velocity of RNAP and dramatically decreased the force where the pause-free velocity is half-maximal (without NusA 18 ± 2 pN of assisting load, with NusA 1 ± 4 pN of opposing load).
Put another way, if load-dependent rate k3 was instead to correspond to tethered-head binding, then an increase in k3 under assisting load would increase the likelihood of completing a step, and thereby increase run lengths in this force regime, contrary to observation.
These data are consistent with the explanation that inter-head tension, which can promote rear-head release, is effectively abolished by extending the NL past its WT length, but that the reduced tension can be restored by applying assisting load, which places differential stress on the rear head.
4) Some additional explanations will be useful on the sentence "…the finding that assisting load fails to appreciably increase the WT velocity suggests that the rate of rear-head release by WT kinesin must be substantially higher than the rate-limiting step(s) of the mechanochemical cycle".
Under assisting loads, only the HsK-CL construct could be sped up beyond WT velocities.
Data were collected under the same conditions as Figure 3. DmK-WT velocity was not affected by assisting loads, but the velocities of all mutant constructs could be increased by larger assisting loads.
In contrast to DmK-WT, which is not sped up by assisting loads, the reduction in unloaded velocity produced by NL extension could be recovered by applying larger assisting loads (∼20 pN).
