RNA polymerase (RNAP) is a DNA-dependent motor protein that links ribonucleotide polymerization to force generation and DNA translocation through its active site, i.e., mechanical work. Single-molecule studies using optical tweezers have allowed researchers to probe the load-dependent ribonucleotide incorporation rate and processivity of both single-subunit viral and multisubunit prokaryotic and eukaryotic RNAPs engaged in transcription elongation. A single-molecule method is described here, which allows the complete transcription cycle (i.e., promoter binding, initiation, elongation and termination) to be followed in real-time using dual-trap optical tweezers and a unique “three-bead” geometry. This single-molecule transcription assay can be used to probe the mechanics of both stationary and moving RNAP–DNA complexes engaged in different stages of transcription.
