Brightness-gated two-color coincidence detection unravels two distinct mechanisms in bacterial protein translation initiation

Commun Biol. 2019 Dec 6:2:459. doi: 10.1038/s42003-019-0709-7. eCollection 2019.

Abstract

Life on the molecular scale is based on a complex interplay of biomolecules under which the ability of binding is crucial. Fluorescence based two-color coincidence detection (TCCD) is commonly used to characterize molecular binding, but suffers from an underestimation of coincident events. Here, we introduce a brightness-gated TCCD which overcomes this limitation and benchmark our approach with two custom-made calibration samples. Applied to a cell-free protein synthesis assay, brightness-gated TCCD unraveled a previously disregarded mode of translation initiation in bacteria.

Keywords: Biochemical assays; Single-molecule biophysics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacteria / genetics*
  • Bacteria / metabolism*
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism*
  • Fluorescence Resonance Energy Transfer
  • Molecular Imaging* / methods
  • Peptide Chain Initiation, Translational*
  • Spectrometry, Fluorescence* / methods

Substances

  • Bacterial Proteins