Cryo-EM of elongating ribosome with EF-Tu•GTP elucidates tRNA proofreading

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA delivered by elongation factor EF-Tu(1). Understanding the molecular mechanism of proofreading requires visualizing GTP-catalyzed elongation, which has remained a challenge(2–4). Here, time-resolved cryo-EM revealed 33 states following aminoacyl-tRNA delivery by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding center (DC) dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis allows EF-Tu’s GTPase domain to extend away, releasing EF-Tu from tRNA. Then, the 30S subunit locks cognate tRNA in the DC, and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase center. By contrast, the DC fails to lock near-cognate tRNA, allowing dissociation of near-cognate tRNA during both initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between initial selection(5,6) and the previously unseen proofreading, which together govern efficient rejection of incorrect tRNA.