Fig. 3: Integrating kinetic and structural studies of translocation.

Top, translocation scheme based on the crystal structure of the intermediate translocation complex reported here (in frame) and on cryo-EM structures of late translocation (PDB ID: 6GZ3 and 6GZ5), as well as hybrid and classical post-translocation states (PDB ID: 3J77 and 3J78). A proposed sequence of events based on kinetic studies²⁸ is shown at the bottom. Steps 1 and 2, thermally driven intersubunit rotations lead to tRNAs adopting hybrid A/P and P/E states and eEF2–GTP binding to the 80S ribosome. Steps 2 and 3, concomitant changes of LSU H69 composing intersubunit bridge B2a and the decoding centre, and insertion of the eEF2 diphthamide to the SSU A-site induce unlocking of the decoding centre. The released codon–anticodon duplex becomes stabilized by direct interactions with diphthamide. Detachment of tRNA ASLs from the SSU body and further insertion of the eEF2 domain IV into the A-site cause initial anticlockwise rotation of the head and movement of the second tRNA from the SSU P-site towards the E-site where it binds to L1 stalk. Steps 3 and 4, eEF2 remains anchored to LSU via domains I and V but is released from SSU where domain IV uncouples tRNA–mRNA from rearrangements of the SSU body and head. What is perceived as an additional large swivelling of the head is actually a result of the body back-rotation while the head remains fixed to tRNAs. Step 4 and 5, this body rotation increases the strain in the SSU neck and leads to uncoupling of the head from tRNAs. Formation of contacts between rRNA of the head and domain IV of eEF2 restrain the head position. The last steps of translocation are achieved when the head, owing to the increasing strain on the neck, snaps back to a non-rotated state and tRNA–mRNA binds to the SSU P-site environment.