Probing Mechanical Flexibility and Discrete Stepping of an Ultra-Fast Ring ATPase
DNA translocation is a fundamental process in biology, required for essential cellular processes including recombination, replication, and chromosome segregation. The SpoIIIE motor is a homo-hexameric dsDNA translocase, part of the ASCE [Additional Strand Conserved E (glutamate)] superfamily of oligomeric ring NTPases. SpoIIIE is found in the bacterium Bacillus subtilis, its biological role is to actively translocate DNA to ensure the proper segregation of sister chromatids. SpoIIIE accomplishes this task by coupling the chemical energy provided by ATP to generate mechanical work, pumping the DNA across a cellular membrane at a rate of 4 kbp/s. While generating mechanical work is a common function of ring NTPases, the timing and order of individual power strokes applies this work in a nuanced manner to better suit particular biological tasks. Understanding the mechanical aspects of fast DNA translocation provides a unique insight into the diverse mechanical strategies employed by these large class of ring NTPases.
Using single-molecule optical trapping techniques, I present real-time measurements of SpoIIIE DNA translocation. By challenging the motor with different lengths of modified DNA substrates, I have determined that SpoIIIE makes critical phosphate contacts with the DNA backbone during translocation and characterized the periodicity of motor-DNA interactions, suggesting a fundamental step size of 2 bp. Furthermore, velocity dependence on an applied external load and various concentrations of ATP, ADP and Pi have demonstrated that translocation is coupled to phosphate release. Finally, ATP analogue experiments are presenting an emerging model of a unique, partially coordinated mechanism of hydrolysis between the individual subunits of SpoIIIE.