Single-Molecule DNA Nanomanipulation: Detection of Promoter-Unwinding Events by RNA Polymerase

This chapter aims to describe a nanomanipulation technique that makes it possible to mechanically and quantitatively stretch and supercoil a single linear DNA molecule. This chapter reviews the technique, which is an extension to the study of protein DNA interactions that leads to DNA untwisting, particularly to the study of promoter unwinding by RNA polymerase during the initiation of transcription. It discusses the response of the DNA to mechanical changes in supercoiling when calibrated, the system can be used to observe in real-time interactions between a single enzyme molecule and its supercoiled DNA substrate. It explains the application of this method in the study of RNA polymerase-induced promoter unwinding, along with bending/compaction of the DNA by RNA polymerase. With this assay, there is a possibility to quantitatively study the role of DNA sequence and supercoiling, temperature, nucleotides, effectors, and activators in formation of the transcription bubble. This technique may be applicable to the real-time study of a variety of protein–DNA interactions that cause deformation of the DNA, such as the unwinding of origins of replication or the binding of transcription factors.