BIOPHYSICS SEMINAR <br> Detection and Manipulation of Single Biomolecular Machines

After twenty-five years of development, single-molecule techniques have become popular tools to study biology. There are two broad families of single-molecule approaches: fluorescence-based detection and force-based manipulation. I will present two systems to demonstrate the power of these approaches in dissecting the molecular mechanism of complex biological processes. First, optical trapping experiments showed that the DNA packaging motor of bacteriophage phi29 simultaneously rotates and translocates DNA. This homomeric ring ATPase displays a remarkable level of coordination and an unexpected division of labor among its subunits. As DNA is packed into the viral capsid, the motor gradually changes its step size and the amount of DNA rotation to cope with the increasing internal pressure, while still remaining highly coordinated. Second, single-molecule FRET assays revealed that the HIV reverse transcriptase is able to flip and slide on nucleic acid substrates. These large-scale motions allow the enzyme to quickly switch between various binding modes and efficiently perform different functions.