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 ribosome•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.
- MeSH
- aminoacyl-tRNA metabolismus MeSH
- elektronová kryomikroskopie * MeSH
- elongační faktor G chemie metabolismus MeSH
- Escherichia coli chemie metabolismus MeSH
- fosfáty metabolismus MeSH
- guanosintrifosfát chemie metabolismus MeSH
- malé podjednotky ribozomu bakteriální chemie metabolismus MeSH
- messenger RNA metabolismus MeSH
- proteosyntéza MeSH
- ribozomy chemie metabolismus MeSH
- RNA transferová metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
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-G•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.
- MeSH
- biokatalýza MeSH
- elektronová kryomikroskopie MeSH
- elongace translace peptidového řetězce genetika MeSH
- elongační faktor G chemie genetika metabolismus MeSH
- Escherichia coli genetika metabolismus MeSH
- konformace nukleové kyseliny MeSH
- konformace proteinů MeSH
- messenger RNA chemie genetika metabolismus MeSH
- molekulární modely MeSH
- posun čtecího rámce na ribozómech genetika MeSH
- proteiny z Escherichia coli chemie genetika metabolismus MeSH
- ribozomy genetika metabolismus ultrastruktura MeSH
- RNA ribozomální 16S chemie genetika metabolismus MeSH
- RNA transferová chemie genetika metabolismus MeSH
- tRNA-methyltransferasy genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH