Ribosomes stalled during translation must be rescued to replenish the pool of translation-competent ribosomal subunits. Bacterial alternative rescue factor B (ArfB) releases nascent peptides from ribosomes stalled on mRNAs truncated at the A site, allowing ribosome recycling. Prior structural work revealed that ArfB recognizes such ribosomes by inserting its C-terminal α-helix into the vacant mRNA tunnel. In this work, we report that ArfB can efficiently recognize a wider range of mRNA substrates, including longer mRNAs that extend beyond the A-site codon. Single-particle cryo-EM unveils that ArfB employs two modes of function depending on the mRNA length. ArfB acts as a monomer to accommodate a shorter mRNA in the ribosomal A site. By contrast, longer mRNAs are displaced from the mRNA tunnel by more than 20 Å and are stabilized in the intersubunit space by dimeric ArfB. Uncovering distinct modes of ArfB function resolves conflicting biochemical and structural studies, and may lead to re-examination of other ribosome rescue pathways, whose functions depend on mRNA lengths.

• MeSH
• Biocatalysis MeSH
• Models, Biological MeSH
• Dimerization MeSH
• Protein Conformation MeSH
• RNA, Messenger genetics   metabolism   ultrastructure   MeSH
• Ribosome Subunits metabolism   MeSH
• Escherichia coli Proteins chemistry   metabolism   ultrastructure   MeSH
• Ribosomes metabolism   ultrastructure   MeSH
• RNA Stability MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• RNA, Messenger MeSH
• Escherichia coli Proteins MeSH

Ribosomes accurately decode mRNA by proofreading each aminoacyl-tRNA that is delivered by the elongation factor EF-Tu1. To understand the molecular mechanism of this proofreading step it is necessary to visualize GTP-catalysed elongation, which has remained a challenge2-4. Here we use time-resolved cryogenic electron microscopy to reveal 33 ribosomal states after the delivery of aminoacyl-tRNA by EF-Tu•GTP. Instead of locking cognate tRNA upon initial recognition, the ribosomal decoding centre dynamically monitors codon-anticodon interactions before and after GTP hydrolysis. GTP hydrolysis enables the GTPase domain of EF-Tu to extend away, releasing EF-Tu from tRNA. The 30S subunit then locks cognate tRNA in the decoding centre and rotates, enabling the tRNA to bypass 50S protrusions during accommodation into the peptidyl transferase centre. By contrast, the decoding centre fails to lock near-cognate tRNA, enabling the dissociation of near-cognate tRNA both during initial selection (before GTP hydrolysis) and proofreading (after GTP hydrolysis). These findings reveal structural similarity between ribosomes in initial selection states5,6 and in proofreading states, which together govern the efficient rejection of incorrect tRNA.

• MeSH
• Cryoelectron Microscopy * MeSH
• Peptide Elongation Factor Tu chemistry   metabolism   ultrastructure   MeSH
• Escherichia coli MeSH
• GTP Phosphohydrolases metabolism   MeSH
• Guanosine Diphosphate chemistry   metabolism   MeSH
• Guanosine Triphosphate chemistry   metabolism   MeSH
• Hydrolysis MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Models, Molecular MeSH
• Ribosomes chemistry   metabolism   ultrastructure   MeSH
• RNA, Transfer chemistry   genetics   metabolism   ultrastructure   MeSH
• Rotation MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Peptide Elongation Factor Tu MeSH
• GTP Phosphohydrolases MeSH
• Guanosine Diphosphate MeSH
• Guanosine Triphosphate MeSH
• RNA, Messenger MeSH
• RNA, Transfer MeSH