Biophysical Society Thematic Meeting| Lima 2019

Revisiting the Central Dogma of Molecular Biology at the Single-Molecule Level

Thursday Speaker Abstracts

CHALLENGING A VIRAL DNA PACKAGING MOTOR WITH MODIFIED SUBSTRATES Juan P. Castillo 1 ; Alexander Tong 1 ; Sara Tafoya 1 ; Paul Jardine 2 ; Carlos Bustamante 1 ; 1 University of California Berkeley, Berkeley, CA, USA 2 University of Minnesota, Minneapolis, MN, USA The DNA packaging motor of the bacteriophage phi29 is a powerful molecular machine that couples the free energy of ATP hydrolysis to DNA translocation. This motor is composed by a pentameric ring ATPase that follows a dwell-burst scheme. In each turn of the mechanochemical cycle, ADP is exchanged for ATP during the dwell time, followed by a translocation burst that is 10 base pairs (bp) in size, which is composed of four consecutive sub-steps of 2.5 bp. Several models can explain what determines the burst size of the motor: the B-form DNA has 10.5 bp per turn of the double helix, suggesting that the structure of the substrate is the determining factor; however, the non-integer nature of the sub-steps during the burst suggests that is the fixed conformational change of the ATPase 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. To test the above hypotheses we challenged the phi29 DNA packaging motor with different substrates bearing the A-form of nucleic acids, using high resolution optical tweezers assay. Our results show indeed that the motor is able to adapt its operation to translocate these different substrates by reducing the size of the burst such that it follows the new helical pitch. We propose a mechanistic model where the motor establishes a critical contact with the substrate at every turn of the double helix during subsequent dwells. Such event is a strong interaction that interrupts the last power stroke during the burst when the motor packages A-form substrates, and it serves as a resetting point for the ring ATPase to complete the turnover cycle.

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