ATP-dependent nucleosome repositioning by chromatin remodeling enzymes requires the translocation of these enzymes along the nucleosomal DNA. Using a fluorescence stopped-flow assay we monitored DNA translocation by a minimal RSC motor and through global analysis of these time courses we have determined that this motor has a macroscopic translocation rate of 2.9 bp/s with a step size of 1.24 bp. From the complementary quantitative analysis of the associated time courses of ATP consumption during DNA translocation we have determined that this motor has an efficiency of 3.0 ATP/bp, which is slightly less that the efficiency observed for several genetically related DNA helicases and which likely results from random pausing by the motor during translocation. Nevertheless, this motor is able to exert enough force during translocation to displace streptavidin from biotinylated DNA. Taken together these results are the necessary first step for quantifying both the role of DNA translocation in nucleosome repositioning by RSC and the efficiency at which RSC couples ATP binding and hydrolysis to nucleosome repositioning.
Bibliographical noteFunding Information:
The authors would like to thank Gada Al-Ani, Koan Briggs, and Micheal Conner for helpful discussion concerning the results and comments on the manuscript. This work was supported by National Institutes of Health Grant P20 RR017708 and by the University of Kansas General Research Fund.
This work was supported by National Institutes of Health Grant P20 RR017708.
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- Chromatin remodeling enzyme
- DNA translocation
- Energy transduction
- Kinetic modelling