Extended Data Fig. 6: Quantification of the intensity of polysomes, ribosome occupancy, and translation elongation.

a, Germplasm of an embryo expressing suntag-nanos flies with SunTag detected by anti-GCN4. The polysomes (arrows) have stronger fluorescence intensities and are co-localized with the mRNA signal (not shown). Individual fully synthesized SunTag-Nanos proteins (examples pointed out by arrowheads) have lower intensities and are not co-localized with mRNA. b, Fluorescence intensities of polysomes and single SunTag-Nanos protein, extracted from FISH-Quant analysis (see Methods). Data from five embryos, each represented by a different color, are plotted as a super-plot. Color-filled circles represent the mean values of the embryos. c, Calculated ribosome occupancy on suntag-nanos mRNA using data from b. Data are the mean ± s.d. d, Theoretical process of fluorescence recovery after photo-bleaching (FRAP). Before photo-bleaching, suntag-nanos mRNA is translated at a steady state with SunTag bound by fluorescent scFv-GFP (phase 1). Photo-bleaching diminishes the fluorescence of bound scFv-GFP (phase 2). Newly synthesized SunTag epitopes after photo-bleaching bind fluorescent scFv-GFP, causing fluorescence recovery of the polysome. Assuming a constant elongation rate, the initial phase of recovery is linear (phase 3). When the peptide that contains the first SunTag synthesized post-bleaching leaves polysome, which counteracts the increase of newly synthesized SunTag, the increase of signal starts to slow down (phase 4). When the first ribosome loaded after photo-bleaching finishes the translation, the signal reaches a plateau (phase 5) with the same intensity as before photo-bleaching because all the SunTags are bound by fluorescent scFv-GFP again. e, A hypothetical FRAP curve (top) based on the theoretical FRAP process, and the FRAP experimental data (bottom, same as Fig. 3e, Data are the mean ± s.d.), which shows a similar curve as the theoretical curve.

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