Fig 1.
Bacterial ribosome and EF-G structures and translocation scheme.
(a) EF-G domains I–V are indicated. This structural model is based on Protein Data Bank (PDB) ID code 4V7B (Ref. 9). (b) EF-G–bound, FA-stalled PRE complex showing compacted positions of deacylated and peptidyl-tRNAs (orange). The rRNA is shown in gray, the ribosomal proteins are shown in blue. The GTPase activating center (GAC) and EF-G are indicated. The structural model is based on PDB ID code 4W29 (Ref. 8). (c) Framework of ribosome translocation in which EF-G(GTP) binds preferentially to PRE complexes in which the small subunit (blue) has rotated with respect to the large subunit (dark gray). EF-G–catalyzed GTP hydrolysis induces “unlocking”, a process that allows the mRNA and tRNA substrates to move relative to the small subunit. Unlocking promotes the (mostly irreversible) formation of the INT2 complex, characterized by tRNA compaction, ~15°–17° head swivel (blue), and partial small subunit back-rotation (light gray). Further (~23°–26°) head swivel (purple), together with complete small subunit back-rotation (white) lead to the (reversible) formation of the INT3 state. Recognition of peptidyl tRNA in the small subunit P site stabilizes INT3 and prompts reverse swivel of the head domain, returning it to its original position (white), from which EF-G(GDP) dissociates.
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).
Fig 3.
Variant helical axis and hinge motions.
(a) Calculated helical axis (red) for the structural core of the 30S head in 16S rRNA. The head (domain III) is shown in cyan, and the body (domains I, II and IV) is shown in green (PDB ID 4V7B, Reference 9). (b) Displacement at each position along the calculated helical axis from the classical PRE state to the simulated conformation at 16° swivel for wild type and top selected hinge 1 variants. (c) Same as (b) for wild type and top selected hinge 2 variants. (d) Hinge 1 motion in the wild type ribosome. Hinge 1 is positioned in a weak point in h28 and its motion results from the kinked helix h28 being straightened during swivel. Cylinders show helical axes in the 16S rRNA core for the classical PRE (tan) and swiveled INT2 (magenta) conformations. (e) Root-mean-square-fluctuations (RMSF) of hinge 1 during forward swivel for wild type and top selected hinge 1 variants. (f) Same as (e) for wild type and top selected hinge 2 variants. Symbol keys are ordered according to their RISE selection enrichment.
Fig 4.
Alternate translocation trajectories of head domain motion.
(a) The main head domain motions during translocation. Solvent view of the 30S subunit (Left) showing head swivel in the direction of translocation. 70S particle (Right) showing the direction of head tilt, orthogonal to head swivel. (b) Relative free energy (ΔG*) landscapes of the forward swivel (PRE to INT3) stage of translocation with head domain swivel and tilt angles as reaction coordinates. Heat maps are obtained by normalizing the number of frames in the targeted MD simulation, where the frame count is divided by the maximum frame count. Shown are the maps for the wild type and seven representative hinge variants active in vitro and in vivo. Maps correspond to ten trajectories per genotype. (c) Interactions between the small subunit and tRNAs for simulated translocation intermediates. Subunit interface view of the simulated wild type and representative variant conformations at 16° of forward swivel showing tRNA contacts in the A (yellow), P (magenta) and E (teal) sites.
Fig 5.
Alternate translocation trajectories of tRNA motion.
(a) Deacyl tRNA motion during forward swivel was tracked by its distance to the E loop (h23, G691-A695). (b) Relative free energy (ΔG*) landscapes of the forward swivel (PRE to INT3) stage of translocation with head domain swivel and E loop-Deacyl tRNA distance as reaction coordinates. Heat maps are obtained by normalizing the number of frames in the targeted MD simulation, where the frame count is divided by the maximum frame count. Shown are the maps for the wild type and seven representative hinge variants active in vitro and in vivo. Maps correspond to ten trajectories per genotype.