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. 2007 Aug 15;104(34):13661–13665. doi: 10.1073/pnas.0705988104

Fig. 1.

Fig. 1.

Schematic of single-molecule FRET experimental setup, examples of resulting FRET traces, and analysis results of cognate (Left) and near-cognate (Right) ternary complex delivery to the ribosome. (A) Single-molecule FRET experimental setup to observe the initial selection of aa-tRNA by the ribosome (8). (B) Typical FRET traces of initial tRNA selection process with the nonhydrolyzable GTP analogue, GDPNP. Materials and methods are explained elsewhere (8). Briefly, the green time trace shows the Cy3 fluorescence intensity, and the orange trace is the Cy5 fluorescence intensity. FRET efficiency is defined as ICy5 /(ICy3 + ICy5). In the presence of GDPNP, aa-tRNA selection process is stalled after the initial selection is complete, thus the system cannot proceed past the mid-FRET state. (C) Postsynchronized histograms and FRET efficiency histograms of cognate and near-cognate delivery of GDPNP stalled ternary complexes (see SI Text). The individual FRET trajectories for the cognate (56 particles) and near-cognate (72 particles) ribosome complexes are superimposed after synchronizing to the first observation of FRET ≥0.27. (Upper) The interaction between the cognate ternary complex and the ribosome results in rapid progression from low- to mid-FRET state, and the majority of near-cognate ribosome complexes retreats from low-FRET state to “0” FRET. (Lower) The mean FRET efficiencies for low- and mid-FRET states in the GDPNP stalled cases. At 15 mM [Mg2+], the mid-FRET efficiencies are 0.433 ± 0.005 and 0.394 ± 0.007 for cognate and near-cognate cases, respectively. The low-FRET efficiencies are 0.330 ± 0.002 and 0.317 ± 0.002 for cognate and near-cognate cases, respectively, showing statistically significant difference in the codon-recognition state. At 5 mM [Mg2+], the mid-FRET efficiencies are 0.438 ± 0.027 and 0.442 ± 0.021 for cognate (59 particles) and near-cognate (55 particles), respectively, and the low-FRET efficiencies are 0.348 ± 0.008 and 0.345 ± 0.004 for cognate and near-cognate cases, respectively (data not shown).