Figure 3.

Figure 3.

Variant ribosomes exhibit altered drug sensitivity. (A,B) BIOLOG phenotypic screening comparing the growth of Δ7prrn-BBB (red) and Δ7prrn-HBB (cyan) in the presence of (A) tetracycline and (B) oxytetracycline. Panels A and B are modified from Kurylo et al. [42]. (C) Schematic of tRNA selection measured using smFRET. FRET is monitored upon delivery of a ternary complex consisting of LD655-labelled Phe-tRNA^Phe in complex with GTP bound elongation factor thermo-unstable (EF-Tu(GTP)) to surface-immobilized initiation complexes containing Cy3-labelled tRNA^fMet in the P site. Incoming aa-tRNA enters the A site through a series of distinct intermediates; codon recognition (CR, FRET = 0.2), GTPase activation (GA, FRET = 0.5) and finally reaches the fully accommodated state (AC, FRET = 0.75). (D) Representative smFRET trace illustrating accommodation of incoming aa-tRNA into the A site through the intermediate steps of tRNA selection. (E,F) Post-synchronized population histograms of smFRET tRNA selection events on BBB and HBB ribosomes isolated from Δ7prrn-BBB and Δ7prrn-HBB respectively, in the presence of (E) tetracycline and (F) oxytetracycline. (G,H) Log odds of incoming Phe-tRNA^Phe reaching the accommodated state in the presence of (G) tetracycline and (H) oxytetracycline. (I) PDB 7N1P was fitted into the head focused-refined cryo-EM maps of the BBB and HBB ribosomes, and the two maps were aligned based on the core of the head domain. The backbone root mean squared deviation (RMSD) between BBB and HBB ribosome structures was calculated in Chimera and the BBB ribosome cryo-EM unsharpened map is coloured according to their backbone deviations. Left and middle panels show two views encompassing the sequence variants, while the right hand panel shows the tetracycline binding site.