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

Kink-turn (K-turn) motifs are asymmetric internal loops found at conserved positions in diverse RNAs, with sharp bends in phosphodiester backbones producing V-shaped structures. Explicit-solvent molecular dynamics simulations were carried out for three K-turns from 23S rRNA, i.e., Kt-38 located at the base of the A-site finger, Kt-42 located at the base of the L7/L12 stalk, and Kt-58 located in domain III, and for the K-turn of human U4 snRNA. The simulations reveal hinge-like K-turn motions on the nanosecond timescale. The first conserved A-minor interaction between the K-turn stems is entirely stable in all simulations. The angle between the helical arms of Kt-38 and Kt-42 is regulated by local variations of the second A-minor (type I) interaction between the stems. Its variability ranges from closed geometries to open ones stabilized by insertion of long-residency waters between adenine and cytosine. The simulated A-minor geometries fully agree with x-ray data. Kt-58 and Kt-U4 exhibit similar elbow-like motions caused by conformational change of the adenosine from the nominally unpaired region. Despite the observed substantial dynamics of K-turns, key tertiary interactions are stable and no sign of unfolding is seen. We suggest that some K-turns are flexible elements mediating large-scale ribosomal motions during the protein synthesis cycle.

• MeSH
• adenin chemie   MeSH
• aminokyselinové motivy MeSH
• biofyzika metody   MeSH
• časové faktory MeSH
• cytosin chemie   MeSH
• elongační faktor G chemie   MeSH
• katalýza MeSH
• konformace nukleové kyseliny MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• makromolekulární látky MeSH
• molekulární konformace MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• oscilometrie MeSH
• párování bází * MeSH
• počítačová simulace MeSH
• rentgenové záření MeSH
• ribozomy chemie   MeSH
• RNA malá jaderná chemie   MeSH
• RNA ribozomální 23S chemie   MeSH
• RNA transferová chemie   MeSH
• RNA chemie   MeSH
• sekundární struktura proteinů MeSH
• sekvence nukleotidů MeSH
• software MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Research Support, U.S. Gov't, P.H.S. MeSH
• Názvy látek
• adenin MeSH
• cytosin MeSH
• elongační faktor G MeSH
• makromolekulární látky MeSH
• RNA malá jaderná MeSH
• RNA ribozomální 23S MeSH
• RNA transferová MeSH
• RNA MeSH
• U4 small nuclear RNA MeSH Prohlížeč