X-ray crystallography study on ribosome recycling: the mechanism of binding and action of RRF on the 50S ribosomal subunit

Abstract

This study presents the crystal structure of domain I of the Escherichia coli ribosome recycling factor (RRF) bound to the Deinococcus radiodurans 50S subunit. The orientation of RRF is consistent with the position determined on a 70S-RRF complex by cryoelectron microscopy (cryo-EM). Alignment, however, requires a rotation of 7 degrees and a shift of the cryo-EM RRF by a complete turn of an alpha-helix, redefining the contacts established with ribosomal components. At 3.3 A resolution, RRF is seen to interact exclusively with ribosomal elements associated with tRNA binding and/or translocation. Furthermore, these results now provide a high-resolution structural description of the conformational changes that were suspected to occur on the 70S-RRF complex, which has implications for the synergistic action of RRF with elongation factor G (EF-G). Specifically, the tip of the universal bridge element H69 is shifted by 20 A toward h44 of the 30S subunit, suggesting that RRF primes the intersubunit bridge B2a for the action of EF-G. Collectively, our data enable a model to be proposed for the dual action of EF-G and RRF during ribosome recycling.

Figure 1

The binding position of domain I of RRF on the 50S subunit. (A) Stereo view of the 2F_o^RRF-DI–F_c^Native electron density map of RRF-DI with the fitted structure of domain I (purple ribbons with cyan side chains) of E. coli RRF (Kim et al, 2000). (B) Sequence alignment of E. coli RRF-DI compared with the full-length E. coli and D. radiodurans RRFs (Swiss-Prot Accession numbers P16174 and Q9RU82, respectively). The regions of α-helical secondary structure are indicated in red. Identical and conservative substitutions are shaded dark and light blue, respectively. The three Gly residues (G) that replace domain II in the RRF-DI protein are in bold typeface, whereas dashes indicate gaps in the protein sequence alignment. A conversion table for the E. coli RRF-DI and full-length RRF can be downloaded from http://www.riboworld.com/pubrel/rrfalign.html. (C) Overall orientation of RRF-DI (purple) on the D. radiodurans 50S subunit. Ribosomal rRNA and proteins are colored gray, except for ribosomal proteins L16 (brown), L11 (cyan) and L27 (pink) and rRNA regions, H69 (orange), H71 (pale blue), H80 (cyan), H93 (yellow) and the H95 (SRL, blue). (D) Superposition of RRF-DI (pink) with positions of A- (green) and P-tRNA (cyan) based on relative positions from 70S·tRNA₃ structure (Yusupov et al, 2001). The docking of the two extremes of domain II (RRF-e1, closed in red and RRF-e2, open in blue) from the NMR analysis of A. aeolicus RRF (Yoshida et al, 2001) is also included, as well as the putative position of a P/E-tRNA hybrid site (orange).

Figure 2

Interactions between domain I of RRF and the 50S subunit. (A) Schematic representation of the interaction of RRF-DI with H69–H71, H80 and H93. The numbering for both E. coli (green) and D. radiodurans (red) is given on the relevant regions of the secondary structure diagram of the 23S rRNA of D. radiodurans (Harms et al, 2001). Arrows indicate hydrogen-bonding distance to the base (square), or backbone interactions with the ribose (pentagon) or phosphate-oxygens (triangle), for each rRNA position. Hydrophobic interactions are indicated with open wedges. The colors of the nucleotides correspond to those presented in (B). (B) Overview of the interactions of RRF-DI with the large ribosomal subunit. Predominant contacts include α3 of RRF-DI with H69 (orange) and position at the base of H71 (green and tan). The relative position of the loop region linking α3 and α4 with H80 (light blue), A2602 in H93 (yellow) and the extensions of ribosomal proteins L16 (brown) and L27 (pink) are illustrated in the background.

Figure 3

Conformational changes induced upon RRF binding to the ribosome. (A) RRF-DI induces a distinct conformation in L27. The native (yellow) and RRF-DI (pink) conformations of the N-terminal region of L27 are shown. Hydrogen bond interactions between Lys4 of L27 and the side chains of Asp145 and Glu147 within the loop between α3 and α4 of RRF-DI (purple) are indicated with dashed green lines. Hydrophobic interactions are present between Lys5 and the N-terminal of α1 and His3 with the α3–α4 loop, including Lys146 of RRF-DI. (B) Movement of A2602 of H93 upon binding of RRF-DI. Comparison of the orientation of A2602 in the RRF-DI D50S structure (orange) with the native (Harms et al, 2001) (purple), CCA-puromycin- (ACCP, green) and sparsomycin-bound (SPAR, yellow) D50S structures (Bashan et al, 2003). Arg154 of α3 of RRF-DI (purple backbone with cyan side chains) forms hydrophobic interactions with A2602 of H93. (C) Binding of RRF-DI induces a shift in the position of H69. Longitudinal view of RRF-DI (purple), with a superposition of H69–H71 from the RRF-DI bound D50S (orange), native D50S (pale blue; Harms et al, 2001) and 70S·tRNA₃ (aqua; Yusupov et al, 2001) structures. The position of h44 (olive) of the 16S rRNA of the small subunit illustrates a potential clash with the position of H69 from the RRF-DI-bound D50S structure.

Figure 4

H69 and H71 are the main interaction partners of RRF-DI. (A) Interactions between α3 and α4 of RRF-DI with H67–H71 of the 23S rRNA. Multiple hydrogen bonds (dashed cyan lines) are formed between α3 (red) and α4 (purple) of RRF-DI with H70(–71) (green) and H71 (pale blue). (B) Interactions between α1 and α3 of RRF-DI with H69 of the 23S rRNA. Arg12 (α1) and Lys138 (α3) form hydrogen bonds (dashed cyan lines) with the backbone of nucleotides C1908 and C1909 of H69 (orange).

Figure 5

Structural model for RRF action on the 70S ribosome. (A) Model for the binding position of RRF on the 70S ribosome. The A. aeolicus RRF structures (Yoshida et al, 2001) containing the most extreme conformations of domain II (RRF-e1, closed conformation in red, and RRF-e2, open conformation in blue, as seen in Figure 1D) were fitted by superimposing domain I with the position of RRF-DI obtained in this study. L11 (cyan), the globular part of S12 (yellow) and h44 of the 16S rRNA (purple) are highlighted, otherwise the 16S and 23S rRNA are colored gray and blue, respectively. The small (30S) and large (50S) subunit ribosomal proteins are colored brown and olive, respectively. (B) The positions of the extremes of domain II in relation to the 30S subunit. Binding of EF-G to the ribosome would force the domain II of RRF-e2 (open, blue) toward the orientation seen in RRF-e1 (closed, red), in closer contact with h44 (purple). Colors of the 30S subunit are as in (A). (C) Model for the interaction between domain II and the hinge region of RRF with domains III and IV of EF-G. The position of EF-G (green) and RRF-e1 (red) are shown. Domain II of RRF is sandwiched between domains III and IV of EF-G (green). H69 (orange) is shown for reference. The gain-of-function mutations, amino-acid substitutions, H504Y and S508F, and deletion of the 4–7 C-terminal residues that restored function to E. coli EF-G in the presence of T. thermophilus RRF (Ito et al, 2002) (blue spheres) are indicated. (D) Overview of the relative orientation of EF-G and the two extreme positions of domain II of RRF. The position of EF-G (green) from the E. coli 70S·EF-G·GDPCP cryo-EM reconstruction (Valle et al, 2003) is docked into the 70S-RRF model, revealing large clashes between domains IV and V of EF-G with domain II of RRF-e2, but less significant overlap is observed between EF-G and the RRF-e1 position of domain II.