Alternate conformational trajectories in ribosome translocation
Fig 2
Head domain hinge variants iterative selection and activity.
(a) Solvent view of the 16S rRNA, showing the head (cyan) and body (green) domains. During translocation the head domain swivels about two rRNA hinge structures. Hinge 1 is a weak point in h28 and hinge 2 is a single-stranded linker between helices h34 and h35. Highlighted in orange and blue are the bases randomized in two separate libraries for hinge 1 and hinge 2, respectively, (PDB ID 4V7B, Reference 9). (b) Head swivel and hinge articulation. During swivel hinge 1 motion results from the kinked h28 being straightened, while the orthogonal hinge 2 motion results from h34 swiveling about h35. Cylinders show helical axes in the 16S rRNA core for the PRE (tan) and INT2, swiveled (magenta) conformations. (c) Iterative selection was carried out using the RISE (Ribosome Synthesis and Evolution) platform. RISE combines an in vitro integrated synthesis assembly and translation (iSAT) system with ribosome display. The platform consists of a library of rRNA variants which are synthesized into ribosome variants in (ribosome-free) S150 cell extract complemented with total ribosome proteins. Functional ribosomes are selected using an expressed tag, and the selected rRNA is amplified and cloned, leading to a new cycle of selection or sequencing. (d) Variant activities in vitro quantified as the fluorescence of sfGFP synthesized in iSAT reactions relative to wild type. Hinge 1 mutants shown in orange, hinge 2 mutants shown in blue (H2_3 and H2_4 could not be cloned). (e) Variant activities in vitro quantified as the fluorescence of sfGFP synthesized in iT reactions (translation with prior ribosome synthesis) relative to wild type. (f) Variant activities in vivo, quantified as the doubling rate of the transformed Squires strains (expressing only the mutant ribosomes) relative to wild type. Wild type cells had a doubling rate of (0.85 ± 0.05) doublings / hour. Error bars correspond to the standard deviation from three independent experiments. The black line denotes wild type activity (= 1).