Mechanistic insights into antibiotic action on the ribosome through single-molecule fluorescence imaging

Single-molecule fluorescence imaging has provided unprecedented access to the dynamics of ribosome function, revealing transient intermediate states that are critical to ribosome activity. Imaging platforms have now been developed that are capable of probing many hundreds of molecules simultaneously at temporal and spatial resolutions approaching the sub-millisecond time and the sub-nanometer scales. These advances enable both steady- and pre-steady state measurements of individual steps in the translation process as well as processive reactions. The data generated using these methods have yielded new, quantitative structural and kinetic insights into ribosomal activity. They have also shed light on the mechanisms of antibiotic targeting the translation apparatus, revealing features of the structure-function relationship that would be difficult to obtain by other means. This review provides an overview of the types of information that can be obtained using such imaging platforms and a blueprint for using the technique to assess how small-molecule antibiotics alter macromolecular functions.