Toxic dipeptide-repeat (DPR) proteins are produced from expanded G4C2 repeats in the C9ORF72 gene, the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥20 repeats inhibit the ribosome's peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryogenic electron microscopy (cryo-EM) reveals that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center (PTC). Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with poly-PR and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

• MeSH
• amyotrofická laterální skleróza * genetika   metabolismus   MeSH
• dipeptidy metabolismus   MeSH
• elektronová kryomikroskopie MeSH
• frontotemporální demence * genetika   metabolismus   MeSH
• lidé MeSH
• protein C9orf72 genetika   metabolismus   MeSH
• proteiny genetika   metabolismus   MeSH
• ribozomy metabolismus   MeSH
• transferasy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• C9orf72 protein, human MeSH Prohlížeč
• dipeptidy MeSH
• protein C9orf72 MeSH
• proteiny MeSH
• transferasy MeSH

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
• Názvy látek
• aminoacyl-tRNA MeSH
• elongační faktor G MeSH
• fosfáty MeSH
• guanosintrifosfát MeSH
• messenger RNA MeSH
• RNA transferová MeSH
• tRNA, peptidyl- MeSH Prohlížeč

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
• Názvy látek
• elongační faktor G MeSH
• messenger RNA MeSH
• proteiny z Escherichia coli MeSH
• RNA ribozomální 16S MeSH
• RNA transferová MeSH
• TrmD protein, E coli MeSH Prohlížeč
• tRNA-methyltransferasy MeSH