Fig. 4.
Postsynchronized, time-resolved population FRET histograms reporting on intermolecular tRNA–tRNA distances within individual ribosomes of an ensemble of particles. Histograms show the evolution of FRET for an ensemble of surface-immobilized 70S complexes carrying tRNAfMet(Cy3-s4U) in the P site after delivery of EF-Tu(GTP)Phe-tRNAPhe(Cy5-acp3U) where individual FRET time traces have been synchronized to the first FRET value ≥ 0.25. Population information is color-coded from tan (lowest occupancy) to red (highest occupancy). The histogram plots generated by stopped-flow delivery of EF-Tu(GTP)Phe-tRNAPhe(Cy5-acp3U) depend on the acylation state of tRNA in the P site and in the presence of EF-G. (A) Complexes carrying fMet-tRNAfMet(Cy3-s4U) in the P site. (B) Complexes carrying OH-tRNAfMet(Cy3-s4U) in the P site. (C) Exactly as in A but in the presence of EF-G. Within experimental uncertainty, the high FRET peaks observed have the same mean FRET value, 0.74 ± 0.05. Similarly, the lower FRET peaks observed in A and B have a mean FRET value of 0.45 ± 0.05. Peaks centered at zero FRET arise from Cy5 blinking, photobleaching, and tRNA dissociation.