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Observing Single RNA Polymerase Molecules Down to Base-Pair Resolution.

Anirban Chakraborty1, Cong A Meng2, Steven M Block3

  • 1Department of Biology, Stanford University, Stanford, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|November 16, 2016
PubMed
Summary
This summary is machine-generated.

This study details optical trapping methods for studying RNA polymerase (RNAP) movement during transcription. Researchers can now analyze both bacterial RNAP and eukaryotic RNAPII with single-base-pair resolution.

Keywords:
Optical trappingRNA polymeraseRNA polymerase IISingle-moleculeTranscription

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Area of Science:

  • Molecular Biology
  • Biophysics

Background:

  • RNA polymerases (RNAP) move via a stepping mechanism during transcription.
  • Optical trapping assays allow real-time monitoring of single RNAP molecules.
  • These assays enable the study of force-dependent transcriptional kinetics.

Purpose of the Study:

  • To describe methods for optical trapping assays of transcriptional elongation.
  • To enable studies on both prokaryotic RNAP and eukaryotic RNAPII.

Main Methods:

  • Reconstitution of transcription elongation complexes.
  • Derivatization of beads for optical trapping.
  • Performing optical trapping measurements on RNAP and RNAPII.

Main Results:

  • Established protocols for optical trapping assays with RNAP and RNAPII.
  • Demonstrated feasibility of single-molecule analysis of transcriptional elongation.

Conclusions:

  • Optical trapping is a powerful technique for dissecting RNAP function.
  • This work extends the application of optical trapping to eukaryotic RNAPII.