Extended Data Fig. 4: An eIF5 FRET signal.
From: eIF1 and eIF5 dynamically control translation start site fidelity
(a) Image of a gel from SDS-PAGE analysis of purified eIF5 labeled on its N-terminal ybbR tag with Cy5 (or Cy5.5) fluorescent dye. The left and right images are total protein and Cy5 fluorescent scans of the same gel. Identical results are obtained with either Cy5 or Cy5.5. (b) Schematic of the equilibrium total internal reflection fluorescence microscopy (TIRFm) experiments. Samples were excited with a 532 nM laser. (c) Example field of view of TIRFm experiments. (d) Example fluorescence data of initiation complexes equilibrated on the model mRNA with an AUG start site. The complexes contain tRNAi-Cy3 (green) to eIF5-Cy5 (purple) FRET events. The top plot represents the fluorescence intensities (arbitrary units) of both signals, and the bottom plot represents the calculated FRET efficiency (EFRET). The right panel plots the EFRET distribution observed during the indicated number of eIF5 binding events. The mean EFRET ± standard deviations are indicated. (e) Cumulative probability plot of the eIF5 FRET lifetime observed on the AUG model mRNA. The line represents a fit to a double-exponential function, which was used to derive the indicated rates.
