Figure 4.
Recent work on bacterial translation involved use of intersubunit dye–quencher labeling (Cy3B-30S and BHQ-2-50S; see Fig. 3C for labeling positions) to track ribosome conformation (green signal). (A) Monitoring Lys-Cy5-tRNALys-binding dynamics (red) on ribosomes with Nm-modified mRNAs (Choi et al. 2018) shows multiple short Cy5 pulses representing tRNA rejection events, explaining the long ribosome stall in nonrotated state. (B) Tracking translation of gene 60 mRNA revealed that the subset of ribosomes that bypassed stalled at the take-off Gly-45 codon in the rotated state. (C) Simultaneously tracking the ribosome intersubunit conformation (green), Cy5-RF binding (red), and P-site tRNA occupancy (Phe-Cy5.5-tRNAPhe, violet) during translation termination and recycling (Prabhakar et al. 2017a) helped resolve the posttermination rotated state, a key intermediate preceding subunit splitting step (catalyzed by RRF and EF-G) and subsequent 30S complex disassembly (performed by IF3).