Protein synthesis takes place in a magnificent macromolecular machine called ribosome in every living cell. The termination of protein synthesis happens when a stop codon is encountered in the ribosome A site. In bacteria, stop codons are recognized by proteins called class I release factors (RF1 and RF2). Upon stop codon recognition, class I release factors catalyze peptide release in the ribosome P site, terminating protein synthesis. After peptide release, a class II release factor (RF3), also a GTPase, promotes the disassociation of the class I release factors from the ribosome. Finally, GTP-hydrolysis on RF3 results in its own disassociation.
We obtained three high-resolution crystal structures of functional complexes of the ribosome bound with release factors along the translational termination pathway. Molecular interactions revealed by these structures, combined with previously available biochemical data, resolve the long-standing questions concerning stop-codon recognition specificity, the catalytic mechanism of peptide release, and the function of RF3 in translational termination. Our results also elucidate structural features of the ratcheted ribosome in the hybrid ligand binding state and provide insights into how a translational GTPase stabilizes the hybrid state of the ribosome in translation.