Figure 3. rrsH-Bearing Ribosomes Are Comparatively Resistant to Tetracycline.
(A) BIOLOG analysis comparing the growth rate of Δ7prrn-HBB (cyan) to that of Δ7prrn-BBB (red) in the presence of tetracycline. Shading indicates SD of three biological replicates.
(B) Schematic of tRNA selection where FRET is monitored between aminoacyl- and peptidyl-tRNA on individual ribosomes. Ternary complex containing an LD650-labeled Phe-tRNAPhe molecule is delivered to surface-immobilized ribosomes programmed with a cognate mRNA codon in the A site and enters the ribosome through distinct intermediate states on pathway to a fully accommodated state where peptide-bond formation occurs.
(C) Representative smFRET trace showing the process of aminoacyl-tRNA entry into the ribosome. Prior to ternary complex binding to the ribosome, no FRET is observed (zero FRET; left). Aminoacyl-tRNA entry into the ribosome occurs through two intermediate states: a low-FRET, codon-recognition (CR) state and an intermediate-FRET, GTPase-activated (GA) state. Transitions between the CR and GA state and the GA to AC (accommodated) state are highlighted. Productive formation of the GA state facilitates entry into the AC state.
(D and E) Post-synchronized, ensemble smFRET histograms of tRNA selection on rrsB-(D) and rrsH-bearing (E) ribosomes in the presence of tetracycline. Plots show the sum of 12 repeats for each experiment and are comprised of >4,000 individual FRET trajectories.
(F) Log odds of tRNA accommodation in the presence of tetracycline (p = 1.2e 20). Error bars indicate SEM and ***p < 0.01 (STAR Methods).