Elongation factor G bound to the ribosome in an intermediate state of translocation

Abstract

A key step of translation by the ribosome is translocation, which involves the movement of messenger RNA (mRNA) and transfer RNA (tRNA) with respect to the ribosome. This allows a new round of protein chain elongation by placing the next mRNA codon in the A site of the 30S subunit. Translocation proceeds through an intermediate state in which the acceptor ends of the tRNAs have moved with respect to the 50S subunit but not the 30S subunit, to form hybrid states. The guanosine triphosphatase (GTPase) elongation factor G (EF-G) catalyzes the subsequent movement of mRNA and tRNA with respect to the 30S subunit. Here, we present a crystal structure at 3 angstrom resolution of the Thermus thermophilus ribosome with a tRNA in the hybrid P/E state bound to EF-G with a GTP analog. The structure provides insights into structural changes that facilitate translocation and suggests a common GTPase mechanism for EF-G and elongation factor Tu.

Fig. 1. Unbiased difference Fourier maps

Unbiased difference Fourier maps obtained after initial refinement with an empty ribosome as a starting model, showing (A) P/E tRNA, (B) switch 1 and GDPCP in the active site, (C-E) key conserved residues in the active site with water molecules.

Fig. 2. EF-G bound to the rotated state of the ribosome

(A) Overall view of EF-G and the ribosome. EF-G is shown in red, the 50S subunit is shown in cyan, the 30S subunit in yellow, the P/E site tRNA in green, and ribosomal protein L1 is shown in orange. (B) Global conformational changes in the 70S ribosome upon GTP hydrolysis as viewed from the perspective of the 23S RNA (cyan). (C) Change in the swivel angle of the head of the 30S in various states of the ribosome, showing the rotated state with EF-G in this study (yellow), the post-translocated state with EF-G and GDP (light gray; pdb code 2WRI) (25), the “TI^Pre” state of a cryoEM structure of EF-G with GPDNP bound to a rotated state (green)(13) and the “TI^Post” state of the same study (red). For clarity, only the ribosomal RNA is shown in B and C.

Fig. 3. Dynamics of the L1 stalk during tRNA translocation

(A) Three distinct conformations of the L1 stalk, showing the open (gray; pdb code 2WA4)(30), the half-closed (pink; pdb code 2WRJ)(25) and fully closed conformations (cyan; this study). (B) The ribosomal protein L1 (orange) stabilizes the distorted P/E tRNA (green) halfway between the canonical P (red) and E (blue, see inset) site conformations. (C) Details of interactions between the L1 protein (orange) and elbow of the P/E tRNA (green). The unbiased F_o-F_c difference Fourier map is contoured at 2.5 σ.

Fig. 4. Interactions of EF-G with L6, L11 and L12

Interactions of EF-G with ribosomal proteins L11, L6, the L12 CTD near the base of the L7/L12 stalk. A single C-terminal domain of L12 is seen to interact with both EF-G and the N-terminal domain of L11.

Fig. 5. Conformational changes in EF-G during translocation

(A) Comparison of isolated EF-G structure (light green; pdb code 2BV3)(36) with EF-G in this study (red). (B) Comparison of EF-G in this study (red) with that in the post-translocated state (gray; pdb code 2WRI)(25) reveals an inter-domain rotation about domain III leading to changes in the orientation of domain IV. Inset (right) shows that in the GDPCP bound EF-G (this study), switch I forms a 3₁₀ helix that stabilizes helix B3 in an altered conformation.

Fig. 6. The active site of EF-G

(A) Sequence alignment of G-domains from several translational GTPases shows conservation of residues Asp22, Lys25 and His87 except in EF-G-2. (B) Details of the catalytic site around the γ-phosphate of GDPCP (blue) with relevant distances displayed as dashes. EF-G residues and waters are in red, Mg⁺⁺ ions in green, and residues of the SRL are in cyan. (C) Comparison of the active site of isolated EF-G with GDPNP (light green; pdb code 2BV3)(36), EF-G with GDPCP in this structure (red) and EF-G in the GDP posttranslocated state (gray; pdb code 2WRI)(25) shows that His⁸⁷ and Asp²² move toward the γ-phosphate of GDPCP on ribosome binding, and away from it upon GTP hydrolysis. (D) Similarity of the activated catalytic sites of EF-G (this structure) and EF-Tu (pdb code 2XQD) (20), suggesting common mechanism of GTPase activation for the two factors.