Single-Molecule Studies of the Temperature Dependence of Viral DNA Packaging Motors

A key step in the assembly of many viruses is the packaging of dsDNA into a preformed capsid by the action of a portal molecular motor complex. ~We have developed methods for directly measuring viral DNA translocation at the single molecule level using optical tweezers and applied these methods to study bacteriophages $\Phi $29, lambda, and T4. Our previous measurements with $\Phi $29 were performed at room temperature. ~Here we report that the rate of DNA translocation is strongly temperature dependent. ~Preliminary measurements indicate that the motor velocity increases $\sim $2-fold, to $\sim $250-300 bp/s when the temperature is increased from $\sim $20 to 30 degrees C. ~As the viral packaging motors are enzymes that catalyze ATP hydrolysis, such a trend with increasing temperature is to be expected, at least up to the point where the motor complex is thermally dissociated or denatured. ~However, the detailed form of the temperature dependence is difficult to quantify using standard bulk assay methods. ~We have installed a heating/cooling system in our optical tweezers instrument that allows us to precisely control the temperature in our sample chamber. ~This system allows us to systematically study the temperature dependence of the DNA translocation rate.