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. 2011 Dec;17(12):2189–2200. doi: 10.1261/rna.029033.111

FIGURE 2.

FIGURE 2.

Compensatory mutations in P- and A-site tRNA CCA ends restore tRNA equilibrium on the pretranslocation ribosome. Cartoons (far left panels) illustrate tRNA dynamics (red stick) on the ribosome, with the 50S (purple) and 30S (orange) subunit. The green and red stars represent the approximate positions of Cy3 and Cy5 fluorophores on tRNA, respectively. The Exit (E), Peptidyl (P), and Aminoacyl (A) sites are labeled below the 30S subunit. (A) Histograms of single-molecule FRET trajectories reveal the tRNA equilibrium at the population level. Left: wild-type (WT) ribosomes with native WT tRNAfMet in the P site and “cut and ligated” WT tRNAPhe in the A site. Center: G2553C ribosomes with native WT tRNAfMet in the P site and “cut and ligated” WT tRNAPhe in the A site. Right: G2553C ribosomes with WT tRNAfMet in the P site and C75G tRNAPhe in the A site. (B) Histograms— Left: WT ribosomes with “cut and ligated” WT tRNAfMet in the P site and native WT tRNAPhe in the A site. Center: G2252C ribosomes with “cut and ligated” WT tRNAfMet in the P site and native WT tRNAPhe in the A site. Right: G2252C ribosomes with C74G tRNAfMet in the P site and native WT tRNAPhe in the A site. The color scale at the bottom of the panel represents the relative populations.