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

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.

(Höfig et al., Commun Biol 2019, 2, 459; doi:10.1038/s42003-019-0709-7)