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Escherichia coli RNA polymerase activity observed using atomic force microscopy

S Kasas1, N H Thomson, B L Smith

  • 1Physics Department, University of California Santa Barbara 93106, USA.

Biochemistry
|January 21, 1997
PubMed
Summary

Atomic force microscopy visualized Escherichia coli RNA polymerase (RNAP) transcription in fluid. This technique revealed variable DNA translocation and transcription rates, offering molecular-level insights into enzyme activity.

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Area of Science:

  • Molecular Biology
  • Biophysics

Background:

  • Atomic force microscopy (AFM) is a powerful tool for visualizing biological molecules.
  • Understanding the dynamics of transcription is crucial for molecular biology.

Purpose of the Study:

  • To observe Escherichia coli RNA polymerase (RNAP) transcription in real-time using fluid tapping-mode AFM.
  • To investigate the translocation of DNA templates by RNAP during transcription.

Main Methods:

  • Fluid tapping-mode atomic force microscopy (AFM) was employed to image RNAP-DNA complexes.
  • Ternary complexes of RNAP, double-stranded DNA (dsDNA), and nascent RNA were adsorbed onto mica and imaged under flowing buffer.
  • Ribonucleoside 5'-triphosphates (NTPs) were introduced to initiate transcription.

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Main Results:

  • AFM imaging revealed variable DNA translocation by RNAP, including DNA being pulled through, dissociating, or remaining stationary.
  • Observed transcription rates ranged from approximately 0.5-2 bases/s at ~5 microM NTP concentrations.
  • While RNA transcripts were not clearly imaged in fluid, successful visualization was achieved using dried samples with a rolling circle DNA template.

Conclusions:

  • Fluid tapping-mode AFM enables real-time observation of molecular-level biological processes like transcription.
  • The study highlights the variability in individual RNAP molecule activity during transcription.
  • AFM provides new insights into the dynamics and mechanisms of transcription at the single-molecule level.