Probing the Mechanics of the Complete DNA Transcription Cycle in Real-Time Using Optical Tweezers
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.
Key wordsSingle molecule Optical trapping T7 RNA polymerase Transcriptional initiation Molecular motor
The authors would like to thank M.L. Bartoo, J.G. Hoggett, J.E. Molloy, A.J. Noël, U. Seger, G.M. Skinner, R. Thieleczek, and C. Veigel for assistance with development of the single-molecule transcription assay, instrument modifications, or data analysis. This work was supported by the BBSRC.
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