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Translation termination involves release factors RF1, RF2 and the GTPase RF3 that recycles RF1 and RF2 from the ribosome. RF3 dissociates from the ribosome in the GDP-bound form and must then exchange GDP for GTP. The 70S ribosome termination complex (70S-TC) accelerates GDP exchange in RF3, suggesting that the 70S-TC can function as the guanine nucleotide exchange factor for RF3. Here, we use cryogenic-electron microscopy to elucidate the mechanism of GDP dissociation from RF3 catalyzed by the Escherichia coli 70S-TC. The non-rotated ribosome bound to RF1 remodels RF3 and induces a peptide flip in the phosphate-binding loop, efficiently ejecting GDP. Binding of GTP allows RF3 to dock at the GTPase center, promoting the dissociation of RF1 from the ribosome. The structures recapitulate the functional cycle of RF3 on the ribosome and uncover the mechanism by which the 70S-TC allosterically dismantles the phosphate-binding groove in RF3, a previously overlooked function of the ribosome., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

During translation, a plethora of protein factors bind to the ribosome and regulate protein synthesis. Many of those factors are guanosine triphosphatases (GTPases), proteins that catalyze the hydrolysis of guanosine 5'-triphosphate (GTP) to promote conformational changes. Despite numerous studies, the function of elongation factor 4 (EF-4/LepA), a highly conserved translational GTPase, has remained elusive. Here, we present the crystal structure at 2.6-Å resolution of the Thermus thermophilus 70S ribosome bound to EF-4 with a nonhydrolyzable GTP analog and A-, P-, and E-site tRNAs. The structure reveals the interactions of EF-4 with the A-site tRNA, including contacts between the C-terminal domain (CTD) of EF-4 and the acceptor helical stem of the tRNA. Remarkably, EF-4 induces a distortion of the A-site tRNA, allowing it to interact simultaneously with EF-4 and the decoding center of the ribosome. The structure provides insights into the tRNA-remodeling function of EF-4 on the ribosome and suggests that the displacement of the CCA-end of the A-site tRNA away from the peptidyl transferase center (PTC) is functionally significant.

Elongation factor 4 (EF4/LepA) is a highly conserved guanosine triphosphatase translation factor. It was shown to promote back-translocation of tRNAs on posttranslocational ribosome complexes and to compete with elongation factor G for interaction with pretranslocational ribosomes, inhibiting the elongation phase of protein synthesis. Here, we report a crystal structure of EF4-guanosine diphosphate bound to the Thermus thermophilus ribosome with a P-site tRNA at 2.9 angstroms resolution. The C-terminal domain of EF4 reaches into the peptidyl transferase center and interacts with the acceptor stem of the peptidyl-tRNA in the P site. The ribosome is in an unusual state of ratcheting with the 30S subunit rotated clockwise relative to the 50S subunit, resulting in a remodeled decoding center. The structure is consistent with EF4 functioning either as a back-translocase or a ribosome sequester., (Copyright © 2014, American Association for the Advancement of Science.)

In bacteria, the hybrid transfer-messenger RNA (tmRNA) rescues ribosomes stalled on defective messenger RNAs (mRNAs). However, certain gram-negative bacteria have evolved proteins that are capable of rescuing stalled ribosomes in a tmRNA-independent manner. Here, we report a 3.2 angstrom-resolution crystal structure of the rescue factor YaeJ bound to the Thermus thermophilus 70S ribosome in complex with the initiator tRNA(i)(fMet) and a short mRNA. The structure reveals that the C-terminal tail of YaeJ functions as a sensor to discriminate between stalled and actively translating ribosomes by binding in the mRNA entry channel downstream of the A site between the head and shoulder of the 30S subunit. This allows the N-terminal globular domain to sample different conformations, so that its conserved GGQ motif is optimally positioned to catalyze the hydrolysis of peptidyl-tRNA. This structure gives insights into the mechanism of YaeJ function and provides a basis for understanding how it rescues stalled ribosomes.