Intersubunit movement is required for ribosomal translocation

Abstract

Translocation of tRNA and mRNA during protein synthesis is believed to be coupled to structural changes in the ribosome. The "ratchet model," based on cryo-EM reconstructions of ribosome complexes, invokes relative movement of the 30S and 50S ribosomal subunits in this process; however, evidence that directly demonstrates a requirement for intersubunit movement during translocation is lacking. To address this problem, we created an intersubunit disulfide cross-link to restrict potential movement. The cross-linked ribosomes were unable to carry out polypeptide synthesis; this inhibition was completely reversed upon reduction of the disulfide bridge. In vitro assays showed that the cross-linked ribosomes were specifically blocked in elongation factor G-dependent translocation. These findings show that intersubunit movement is required for ribosomal translocation, accounting for the universal two-subunit architecture of ribosomes.

Fig. 1.

Position of the intersubunit cross-link. The observed axis of rotation from cryo-EM reconstructions (7) is indicated by a red dot, and the positions of cysteines introduced into proteins S6 (F81C) and L2 (I123C) are indicated by yellow dots in the structures of the 50S subunit interface (A), 30S subunit interface (B), and 70S ribosome (C) (8). The position of EF-G (7) is shown in transparent red with dotted outline, and the A-, P- and E-site tRNAs are in yellow, orange, and red, respectively.

Fig. 2.

Formation of an intersubunit disulfide bridge. (A) Sedimentation analysis of S6-L2 mutant ribosomes under subunit dissociation conditions (1 mM Mg²⁺). Under oxidizing conditions the 30S and 50S subunits are covalently linked by disulfide formation to form 70S particles. The arrow indicates the direction of sedimentation. (B) Identification of the S6(F81C)–L2(I123C) cross-link. Proteins from oxidized mutant 70S ribosomes were analyzed by 2D SDS-gel electrophoresis run under oxidizing conditions in the first dimension and reducing conditions in the second dimension. Asterisk indicates the position of free L2 in the first-dimension gel. The off-diagonal spots confirm disulfide cross-linking of S6 and L2.

Fig. 3.

Effects of intersubunit cross-linking on translational functions. (A) The S6-L2 cross-link blocks in vitro translation of a poly(U) mRNA; activity is restored upon reduction of the disulfide bridge by addition of DTT (arrow). (B) Intersubunit cross-linking does not prevent tRNA binding or peptide bond formation. P-site complexes (P-site) contained m32 mRNA and N-Ac-[³H]Phe-tRNA^Phe. A-site complexes (A-site) contained m32 mRNA, tRNA^fMet bound to the P site, and N-Ac-[³H]Phe-tRNA^Phe in the A site. tRNA binding (to 1.5 pmol of ribosomes) was assayed by filter binding, and peptide bond formation was by formation of N-Ac-[³H]Phe-puromycin under oxidizing (ox) or reducing (red) conditions. N-Ac-Phe-tRNA^Phe is reactive with puromycin when bound to the ribosomal P site and unreactive when in the ribosomal A site. (C) Intersubunit cross-linking does not prevent EF-Tu-dependent binding of aminoacyl-tRNA to the A site. [³H]Phe-tRNA^Phe was added to ribosomes containing m32 mRNA and fMet-tRNA^fMet either as a ternary complex with EF-Tu·GTP (+EF-Tu) or with GTP alone (−EF-Tu). Binding of [³H]Phe-tRNA^Phe was normalized to that of WT ribosomes measured under reducing conditions.

Fig. 4.

Intersubunit cross-linking blocks EF-G-dependent translocation. (A) Monitoring translocation of peptidyl-tRNA into the P site by the appearance of puromycin reactivity. Deacylated tRNA^fMet and N-Ac-[³H]Phe-tRNA^Phe were bound to the P and A sites, respectively, of m32-programmed ribosomes. Formation of N-Ac-[³H]Phe-puromycin was measured before (−EF-G) and after (+EF-G) translocation. WT, S6(F81C) and L2(I123C) single mutants, and S6(F81C)- L2(I123C) double mutant ribosomes were assayed under oxidizing (ox) and reducing (red) conditions. (B) Monitoring translocation by toe-printing. Complexes were assembled as in A except with m291 mRNA. Translocation of mRNA was followed by extending a primer bound to the 3′ side of the mRNA with reverse transcriptase. tRNA was first bound to the P site (P) giving the band at +16. The appearance of doublet bands (A) corresponds to A-site tRNA binding. The appearance of band (+19) in the EF-G (+) lane corresponds to translocation by one codon.