Translation studies with single-molecule resolution.

A) Imaging single-molecule translation dynamics is achieved by the measurement of fluorescence spots that are produced when nascent proteins display epitopes that are recognized by antibody fragments bound to fluorescent probes. The gene construct encodes a 10X FLAG SM tag followed by a protein of interest (POI) and the 24X MS2 tag in the 3’ UTR region. B) Microscopy image showing translation at single-molecule resolution; red spots represent single mRNA, and green spots represent nascent proteins. Below is a representative trace showing the intensity fluctuation dynamics of a single-transcript translating FLAG-10X-KDM5B. C) Simulated time courses representing the characteristic single-molecule fluctuation dynamics. A representative trace is selected and highlighted. At the bottom of the figure is given the normalized auto-covariance function (G(τ)) calculated from simulated time courses. The time at which G(τ) hits zero represents the dwell-time (τ_FCS). D) Harringtonine inhibits the translation initiation step by binding to the ribosomal 60S subunit. The plot shows the average fluorescence after Harringtonine treatment. Without new initiation events, the fluctuations diminish causing the intensity to drop to zero at time τ_ROA. E) FRAP causes a rapid drop in the fluorescent intensity and a subsequent recovery that is proportional to the time needed by ribosomes to produce new nascent proteins with non-photobleached probes. The bottom plot shows the temporal dynamics of FRAP, where it can be observed by the abrupt decrease in intensity and a recovery time (τ_FRAP) correlated with the gene length. All simulations correspond to KDM5B for 100 spots and a frame rate of 1 FPS. Error bars represent the standard error of the mean.