Measuring The Physical Step-Size Of Bacteriophage T7 Dna Helicase With Dual Optical Tweezers

Abstract

Helicases are ubiquitous molecular motors which unwind double stranded DNA into two single strands. T7gp4 is a model hexameric helicase from the replication system of bacteriophage T7. The step size of T7gp4 was probed using single molecule optical trapping experiments. To obtain sufficient resolution over experimental timescales a dual optical trap was constructed, decoupling the experiment from the surface. Numerical simulations of helicase in the experimental configuration were conducted to examine the required measurement bandwidth. A novel method of forming double tethers between the dual traps was implemented. A range of steps of the helicase were observed. In combination with structural data and biochemical measurements, this agrees with a sequential mechanism for T7gp4 translocation.