Engineering Approaches to Biomolecular Motors

Engineering Approaches to Biomolecular Motors: From in vitro to in vivo Wednesday Speaker Abstracts

Mechanical Tension vs. Force: Different Ways to Control the Activities of Molecular Motors Working on DNA. Borja Ibarra 1 , Jose Morin 1 , Francisco J. Cao 2 , Margarita Salas 3 . 1 IMDEA Nanoscience, Madrid, Spain, 2 Universidad Complutense de Madrid, Madrid, Spain, 3 Centro Biologia Molecular-Severo Ochoa (CBMSO-CSIC), Madrid, Spain. Single molecule force spectroscopy approaches have proven useful in studying the real time kinetics and mechano-chemical processes governing the operation of molecular motors. Mechanical force perturbs the interactions of the motor protein with its track and modulates the rates of the steps of the reaction located along the force application coordinate. Using optical tweezers and the multifunctional Phi29 DNA polymerase (a hybrid polymerase-helicase), we show how different pulling geometries on a single polymerase-DNA complex modulate the rate of DNA synthesis by acting on different steps of the reaction cycle: 1. Mechanical tension applied longitudinally along the DNA track modulates the equilibrium between the synthetic (pol) and degradative (exo) activities of the polymerase. Tension promotes the intramolecular transfer of the DNA primer strand from the pol to the exo active sites in a similar way to the incorporation of a mismatched nucleotide. 2. Tension applied to the ends of the complementary strands of a DNA hairpin favours the mechanical unwinding of the DNA duplex and modulates the coupling between the DNA synthetic and unwinding reactions of the polymerase. 3. Mechanical force, or load, applied directly on the polymerase acts specifically on the step of the reaction coupled to directional motion of the protein along the DNA and can be used as a variable to determine the coupling mechanism between chemical and mechanical energy during the DNA replication reaction. Our results reveal that changing the pulling geometries on a single motor-track complex (polymerase-DNA) provides well-defined reaction coordinates to modify and quantify the kinetic rates, equilibrium constants and conformational changes of the steps of the reactions responsible for the motor operation.

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