Fig. 4.

Translation kinetics in live cells measured with single-molecule FRAP of TLS.

(A) Schematics of FRAP. The existing NAPs on the TLS were bleached with a focused 491-nm laser at t = 0 (black). The fluorescence recovers as existing ribosomes synthesize the SunTag motifs and new ribosomes arrive and make new NAPs (green). At t = ∞, the fluorescence should recover to the beginning value at steady state. (B toD) Montage of FRAP experiments at different time points for (B) control, (C) nonbleached TLS, and (D) CHX treatment. (E) There was little recovery for CHX treatment (D) (black squares) compared with control (B) (blue circles). Fitting the theory (red line) to the experimental data (n = 31 movies) yielded the translation elongation speed v = 4.7 ± 0.6 amino acids/s.