Figure 1.

Stalled and ongoing tRNA-L11 FRET measurements. L11(Cy5) ribosomes were programmed with mRNA-6,7 (Table 1) and underwent FRET with Val-tRNA^Val(Cy3). (A) Schematic of classical/hybrid equilibrium of a stalled ribosome. The green and red stars represent Cy3 fluorophore and Cy5 fluorophore, respectively. (B) Representative FRET recording. (C) Frames of fluctuating stalled traces formed a two-peaked distribution fit with a double Gaussian function (n = 592 molecules). (D–F) 2 μM EF-G was present during ongoing translation from codons 1–7. (D) Reaction scheme for ongoing translation. (E) A representative trace with the following FRET states: i) the POST state after the ribosome has translated through the fifth (Tyr) and sixth (Phe) codons, with peptidyl-tRNA^Phe residing in the P site; ii) INT_ongoing; and iii) the POST complex after translocation. (F) Frames of traces during ongoing translation formed a peaked PRE-state distribution fit with a single Gaussian component (n = 3664 molecules).