The biophysics of molecular motors: Optical trapping studies of kinesin and RNA polymerase.

摘要

Molecular motors, tiny protein engines, convert the chemical energy stored in a molecular bond into mechanical work. They are responsible for producing a variety of complex movements within biological cells. In the past two decades, optical trapping techniques have been developed to study the motions of these enzymes with ever increasing precision. Here, we present several optical trapping studies of single molecules of kinesin and RNA polymerase. Kinesin, the enzyme responsible for anterograde vesicle transport, moves along microtubule filaments in discrete eight-nm steps. Using a two-dimensional force clamp, we subjected individual kinesin molecules to a variety of loading and ATP conditions. This data is explained with a five-state, mechanochemical model of kinesin stepping that includes three motion-producing biochemical transitions. RNA polymerase is the enzyme responsible for the transcription of DNA into RNA. Using a new experimental assay of polymerase motion, we provide evidence for a proofreading mechanism in which RNA polymerase is able to correct mistakes in the nascent RNA. In addition, we used an optical trap as a local heater to measure the effect of changes in temperature on RNA polymerase motion. The stepping of any molecular motor is stochastic in nature and is governed by biochemical kinetics. We extend the current theory of this stochasticity to include motors which step along sequences of DNA and motors whose step size is variable.