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, 104 (50), 19671-8

The Process of mRNA-tRNA Translocation

Affiliations

The Process of mRNA-tRNA Translocation

Joachim Frank et al. Proc Natl Acad Sci U S A.

Abstract

In the elongation cycle of translation, translocation is the process that advances the mRNA-tRNA moiety on the ribosome, to allow the next codon to move into the decoding center. New results obtained by cryoelectron microscopy, interpreted in the light of x-ray structures and kinetic data, allow us to develop a model of the molecular events during translocation.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Atomic model of the 30S subunit of the pretranslocational ribosome from Thermus thermophilus (ref. ; Protein Data Bank ID code 2J00). This intersubunit view of the 30S highlights several important features, which are color-coded and labeled. Ribosomal proteins S12 and S13 interact directly with the 50S subunit and affect ratcheting efficiency of the ribosome. The mRNA channel passes between the head and the body of the SSU (mRNA is colored gray). Upon EF-G binding, the top of helix 44 (h44) bends from the A site toward the P site. Because helix 44 is directly connected to helix 28 (h28 or the “neck”), the movement in h44 could provide torsional force on h28. GTP hydrolysis by EF-G decouples the tether between the A-site tRNA bound to the head and the decoding center (DC) in the body of the SSU. This event would release the head to rotate about its neck and relieve the torsional force in h28. The head rotation is likely to be important for movement of the mRNA and tRNA anticodon stem-loops on the SSU.
Fig. 2.
Fig. 2.
Binding of EF-G stabilizes the ratcheted conformation of the ribosome. (a) Cryo-EM reconstruction of the pretranslocational ribosome. The large subunit is colored blue, the small subunit is colored yellow, and the P-site tRNA is colored green. (b) Cryo-EM reconstruction of the EF-G·70S complex, showing EF-G in red. The large subunit of the two reconstructions (a and b) was aligned and the small subunit of each was superimposed (c). This view, from the solvent side of the small subunit, shows that binding of EF-G induces a counterclockwise rotation of the small subunit (pink) when compared with the small subunit of the pretranslocational ribosome (yellow).
Fig. 3.
Fig. 3.
The bridge B1b, formed by S13 and L5, in the two ratchet-related conformations of the ribosome. (a and b) Positions of proteins S13 and L5 in the two states. (c and d) Surface charge representation of S13-L5 in the two states. In c (normal conformation), the charges at the interface are of opposite polarity, leading to stabilization. In d (ratcheted conformation), the charges are of equal polarity, leading to instability.
Fig. 4.
Fig. 4.
The P/E-site tRNA visualized in cryo-EM reconstructions. (a) A side view of the 80S·ADPR-eEF2·GDPNP complex (23), showing the CCA end of the tRNA occupying the E site of the large subunit, while the anticodon stem-loop occupies the P site of the small subunit. The A, P, and E sites of the ribosome are individually labeled. (b) The anticodon stem-loop of the P/E-site tRNA forms a strong interaction with the small subunit head, near the G1338-U1341 ridge, and a weaker interaction with the small subunit body, via the A790 loop of the 18S rRNA. The gap between A790 and A1340 separates the ribosomal P and E sites of the small subunit. (c) Stereo view of the interactions between the small subunit and P/E-site tRNA described in b. The head of the small subunit in this complex is rotated, increasing the distance between A790 in the body and A1339 in the head of the small subunit from ≈18 Å to ≈26 Å. This distance would allow passage of the anticodon stem-loop from the P to the E site; however, the tRNA remains bound in the hybrid state. Passage of the tRNA anticodon stem-loop from the P to the E site of the small subunit therefore must occur during back-ratcheting and back-rotation of the head of the small subunit, once EF-G/eEF2 dissociates from the ribosome. E denotes the E site of the small subunit.
Fig. 5.
Fig. 5.
Proposed sequence of events during the translocation process. Schematic depicts the top view of the translating ribosome. EF-G interacts with the pretranslocational ribosome (a), stabilizing the A- and P-site tRNAs into A/P and P/E hybrid sites, respectively. Binding of EF-G is coincident with the ratcheting motion of the entire small subunit (i.e., head and body ratchet together) with respect to the small subunit (b). GTP hydrolysis causes the tip of domain IV of EF-G to sever the connection between the small subunit head–tRNA–mRNA complex and the small subunit body (c). Once the link to the body is severed, the head rotates, translocating the mRNA by one codon and the anticodon stem-loop ends of the hybrid site tRNAs to the P and E sites (d). Translocation is completed by back-rotation of the head and reverse-ratcheting of the entire small subunit as EF-G leaves the ribosome with the tRNAs in full P/P and E/E sites (e).

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