Rotation Tracking Using DNA Origami Rotors Reveals Single Base Pair Steps During Transcription by RNA Polymerase

Abstract

Protein-DNA reactions such as transcription, editing and repair consist of a series of mechanical movements, yet the vast majority of these minute movements have remained challenging or impossible to measure. We have developed a method to track DNA rotation, origami-rotor-based imaging and tracking (ORBIT), that uses fluorescently labeled nano-rotors to amplify and track the movements of single DNA molecules. We used ORBIT to study transcription and discovered that RNA polymerase (RNAP) rotates DNA in single base pair steps during transcription elongation, revealing a close coupling between the enzyme and the helical structure of the DNA template. Intriguingly, these single base pair rotational steps did not appear identical in magnitude. Rather, they ranged from 25° to 40°, suggesting a heterogeneity of movement that we speculate is caused by the sequence-dependent local geometry of DNA. Consistent with this interpretation, twist angles between subsequent base pairs in B-DNA have been reported to range from 27° to 40° in a sequence-dependent manner. Due to its simplicity and generalizability, we anticipate that ORBIT will have broad applications in illuminating the rotational movements of a wide variety of protein-DNA reactions.