ABSTRACT
During translation, a conserved GTPase elongation factor-EF-G in bacteria or eEF2 in eukaryotes-translocates tRNA and mRNA through the ribosome. EF-G has been proposed to act as a flexible motor that propels tRNA and mRNA movement, as a rigid pawl that biases unidirectional translocation resulting from ribosome rearrangements, or by various combinations of motor- and pawl-like mechanisms. Using time-resolved cryo-EM, we visualized GTP-catalyzed translocation without inhibitors, capturing elusive structures of ribosomeâ¢EF-G intermediates at near-atomic resolution. Prior to translocation, EF-G binds near peptidyl-tRNA, while the rotated 30S subunit stabilizes the EF-G GTPase center. Reverse 30S rotation releases Pi and translocates peptidyl-tRNA and EF-G by ~20 Å. An additional 4-Å translocation initiates EF-G dissociation from a transient ribosome state with highly swiveled 30S head. The structures visualize how nearly rigid EF-G rectifies inherent and spontaneous ribosomal dynamics into tRNA-mRNA translocation, whereas GTP hydrolysis and Pi release drive EF-G dissociation.
Subject(s)
Cryoelectron Microscopy , Guanosine Triphosphate/chemistry , Peptide Elongation Factor G/chemistry , Ribosomes/chemistry , Escherichia coli/chemistry , Escherichia coli/metabolism , Guanosine Triphosphate/metabolism , Peptide Elongation Factor G/metabolism , Phosphates/metabolism , Protein Binding , Protein Biosynthesis , RNA, Messenger/metabolism , RNA, Transfer/metabolism , RNA, Transfer, Amino Acyl/metabolism , Ribosome Subunits, Small, Bacterial/chemistry , Ribosome Subunits, Small, Bacterial/metabolism , Ribosomes/metabolismABSTRACT
Frameshifting of mRNA during translation provides a strategy to expand the coding repertoire of cells and viruses. How and where in the elongation cycle +1-frameshifting occurs remains poorly understood. We describe seven ~3.5-Å-resolution cryo-EM structures of 70S ribosome complexes, allowing visualization of elongation and translocation by the GTPase elongation factor G (EF-G). Four structures with a + 1-frameshifting-prone mRNA reveal that frameshifting takes place during translocation of tRNA and mRNA. Prior to EF-G binding, the pre-translocation complex features an in-frame tRNA-mRNA pairing in the A site. In the partially translocated structure with EF-Gâ¢GDPCP, the tRNA shifts to the +1-frame near the P site, rendering the freed mRNA base to bulge between the P and E sites and to stack on the 16S rRNA nucleotide G926. The ribosome remains frameshifted in the nearly post-translocation state. Our findings demonstrate that the ribosome and EF-G cooperate to induce +1 frameshifting during tRNA-mRNA translocation.
Subject(s)
Frameshifting, Ribosomal/genetics , Peptide Chain Elongation, Translational/genetics , Peptide Elongation Factor G/genetics , RNA, Messenger/genetics , RNA, Transfer/genetics , Ribosomes/genetics , Biocatalysis , Cryoelectron Microscopy , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Models, Molecular , Nucleic Acid Conformation , Peptide Elongation Factor G/chemistry , Peptide Elongation Factor G/metabolism , Protein Conformation , RNA, Messenger/chemistry , RNA, Messenger/metabolism , RNA, Ribosomal, 16S/chemistry , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 16S/metabolism , RNA, Transfer/chemistry , RNA, Transfer/metabolism , Ribosomes/metabolism , Ribosomes/ultrastructure , tRNA Methyltransferases/genetics , tRNA Methyltransferases/metabolismABSTRACT
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.
