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. 2017 Dec 5;113(11):2326–2335. doi: 10.1016/j.bpj.2017.08.052

Figure 2.

Figure 2

Stalled and ongoing tRNA-tRNA FRET measurements. Unlabeled ribosomes were programmed with mRNA-6,7 (Table 1) and Phe-tRNAPhe(Cy5) at codon 6 underwent FRET with Val-tRNAVal(Cy3) at codon 7. (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 = 531 molecules). (D–F) 2 μM EF-G was present during ongoing translation from codons 1–7. (D) Reaction scheme for ongoing translation. The yellow triangle represents EF-G; the purple disc represents EF-Tu. (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-tRNAPhe residing in the P site; (ii, iii) INTongoing and the initial POST state, which have similar FRET efficiencies (21); and iv) the POST state after dissociation of tRNAPhe(Cy5) from the E-site. (F) Frames of traces during ongoing translation formed a peaked PRE state distribution fit with a single Gaussian component (n = 2359 molecules).