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Probing the Mechanics of the Complete DNA Transcription Cycle in Real-Time Using Optical Tweezers

  • Christoph G. BaumannEmail author
  • Stephen J. Cross
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 778)

Abstract

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 words

Single molecule Optical trapping T7 RNA polymerase Transcriptional initiation Molecular motor 

Notes

Acknowledgments

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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Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Department of BiologyUniversity of YorkYorkUK

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