Challenging a DNA Packaging Motor with a Modified Substrate

Abstract

The DNA packaging motor of the bacteriophage phi29 is a powerful molecular machine that couples the free energy of ATP hydrolysis with DNA translocation in order to complete the production of new viral particles. The active part of this motor is a pentameric ring ATPase which mechanochemical cycle has been described with exquisite detail using hi-resolution optical tweezers. It was described that in each turn of the cycle, the motor packages DNA taking discrete steps of 10 base pairs (bp) each, in what is called a burst of translocation. At the same time it was shown that this 10 bp burst is composed of four 2.5 bp sub-steps, presumably reflecting the power stroke of the individual ATPases. Several models can explain what is the origin of the burst size: the helical pitch of B-form DNA is 10.5 bp/turn of the double helix, suggesting that the structure of the substrate is what determines the burst size; however, the non-integer nature of the sub-steps within the burst allows to hypothesize that is the ATPase’s conformational change what sets the burst size. Yet another possibility is that the DNA packaging motor switches the local conformation of the DNA substrate from B-form to A-form during packaging (DNA scrunching). To test the above hypotheses we challenged the phi29 DNA packaging motor with a double-stranded RNA substrate (that adopts the A-form of nucleic acids) and measured the packaging activity using optical tweezers.