The ribosome as a molecular machine: the mechanism of tRNA–mRNA movement in translocation

2011 ◽  
Vol 39 (2) ◽  
pp. 658-662 ◽  
Author(s):  
Marina V. Rodnina ◽  
Wolfgang Wintermeyer
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Thermal Fluctuations ◽  
Spontaneous Movement ◽  
Conformational States ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Directional Bias ◽  
Loosely Coupled ◽  
Dynamic Events

Translocation of tRNA and mRNA through the ribosome is one of the most dynamic events during protein synthesis. In the cell, translocation is catalysed by EF-G (elongation factor G) and driven by GTP hydrolysis. Major unresolved questions are: how the movement is induced and what the moving parts of the ribosome are. Recent progress in time-resolved cryoelectron microscopy revealed trajectories of tRNA movement through the ribosome. Driven by thermal fluctuations, the ribosome spontaneously samples a large number of conformational states. The spontaneous movement of tRNAs through the ribosome is loosely coupled to the motions within the ribosome. EF-G stabilizes conformational states prone to translocation and promotes a conformational rearrangement of the ribosome (unlocking) that accelerates the rate-limiting step of translocation: the movement of the tRNA anticodons on the small ribosomal subunit. EF-G acts as a Brownian ratchet providing directional bias for movement at the cost of GTP hydrolysis.

scholarly journals Structural mechanism of GTPase-powered ribosome-tRNA movement

2021 ◽  
Author(s):  
Valentyn Petrychenko ◽  
Bee-Zen Peng ◽  
Ana C. de A. P. Schwarzer ◽  
Frank Peske ◽  
Marina V. Rodnina ◽  
...  
Keyword(s):  
Large Scale ◽  
Ribosomal Subunit ◽  
Molecular Switches ◽  
Thermal Fluctuations ◽  
23S Rrna ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Structural Mechanism ◽  
Molecular Movement ◽  
Switch Regions

GTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP—Pi form stabilizes ribosomal subunit rotation and induces twisting of the sarcin-ricin loop of the 23S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a cascade of rearrangements and a large-scale rotation of EF-G that exerts force on the ribosome and ultimately drives tRNA movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

scholarly journals Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Christine E. Carbone ◽  
Anna B. Loveland ◽  
Howard B. Gamper ◽  
Ya-Ming Hou ◽  
Gabriel Demo ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Atomic Resolution ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
30S Subunit

AbstractDuring 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.

Crystallographic studies of elongation factor G

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1209-1216 ◽  
Author(s):  
Anders Liljas ◽  
Arnthor Ævarsson ◽  
Salam Al-Karadaghi ◽  
Maria Garber ◽  
Julia Zheltonosova ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Change ◽  
Conformational Changes ◽  
Elongation Factor ◽  
Elongation Factors ◽  
Elongation Factor G ◽  
Conformational States ◽  
Gtp Hydrolysis ◽  
Large Conformational Change ◽  
Factor G

The elongation factors G (EF-G) and Tu (EF-Tu) go through a number of conformation states in their functional cycles. Since they both are GTPases, have similar G domains and domains II, and have similar interactions with the nucleotides, then GTP hydrolysis must occur in similar ways. The crystal structures of two conformational states are known for EF-G and three are known for EF-Tu. The conformations of EF-G∙GDP and EF-Tu∙GTP are closely related. EF-Tu goes through a large conformational change upon GTP cleavage. This conformational change is to a large extent due to an altered interaction between the G domain and domains II and III. A number of kirromycin-resistant mutations are situated at the interface between domains I and III. The interface between the G domain and domain V in EF-G corresponds with this dynamic interface in EF-Tu. The contact area in EF-G is small and dominated by interactions between charged amino acids, which are part of a system that is observed to undergo conformational changes. Furthermore, a number of fusidic acid resistant mutants have been identified in this area. All of this evidence makes it likely that EF-G undergoes a large conformational change in its functional cycle. If the structures and conformational states of the elongation factors are related to a scheme in which the ribosome oscillates between two conformations, the pretranslocational and posttranslocational states, a model is arrived at in which EF-Tu drives the reaction in one direction and EF-G in the opposite. This may lead to the consequence that the GTP state of one factor is similar to the GDP state of the other. At the GTP hydrolysis state, the structures of the factors will be close to superimposable.Key words: elongation factor G, elongation factor Tu, crystal structures, conformational changes, ribosomal conformation.

scholarly journals Time-resolved cryo-EM visualizes ribosomal translocation with EF-G and GTP

2021 ◽  
Author(s):  
Christine E Carbone ◽  
Anna B Loveland ◽  
Howard Gamper ◽  
Ya-Ming Hou ◽  
Gabriel Demo ◽  
...  
Keyword(s):  
Elongation Factor ◽  
Atomic Resolution ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
30S Subunit

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.

scholarly journals Elongation factor-Tu can repetitively engage aminoacyl-tRNA within the ribosome during the proofreading stage of tRNA selection

2020 ◽  
Vol 117 (7) ◽  
pp. 3610-3620 ◽  
Author(s):  
Justin C. Morse ◽  
Dylan Girodat ◽  
Benjamin J. Burnett ◽  
Mikael Holm ◽  
Roger B. Altman ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Ternary Complex ◽  
Bond Formation ◽  
Critical Role ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Elongation Factor Tu ◽  
Peptide Bond Formation ◽  
Gtp Hydrolysis

The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tu⋅GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tu⋅GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.

scholarly journals Structural mechanism of GTPase-powered ribosome-tRNA movement

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Valentyn Petrychenko ◽  
Bee-Zen Peng ◽  
Ana C. de A. P. Schwarzer ◽  
Frank Peske ◽  
Marina V. Rodnina ◽  
...  
Keyword(s):  
Large Scale ◽  
Thermal Fluctuations ◽  
Small Subunit ◽  
Forward Movement ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Ribosomal Subunits ◽  
Structural Mechanism ◽  
Molecular Movement ◽  
Switch Regions

AbstractGTPases are regulators of cell signaling acting as molecular switches. The translational GTPase EF-G stands out, as it uses GTP hydrolysis to generate force and promote the movement of the ribosome along the mRNA. The key unresolved question is how GTP hydrolysis drives molecular movement. Here, we visualize the GTPase-powered step of ongoing translocation by time-resolved cryo-EM. EF-G in the active GDP–Pi form stabilizes the rotated conformation of ribosomal subunits and induces twisting of the sarcin-ricin loop of the 23 S rRNA. Refolding of the GTPase switch regions upon Pi release initiates a large-scale rigid-body rotation of EF-G pivoting around the sarcin-ricin loop that facilitates back rotation of the ribosomal subunits and forward swiveling of the head domain of the small subunit, ultimately driving tRNA forward movement. The findings demonstrate how a GTPase orchestrates spontaneous thermal fluctuations of a large RNA-protein complex into force-generating molecular movement.

scholarly journals The Archaeal Elongation Factor EF-2 Induces the Release of aIF6 From 50S Ribosomal Subunit

2021 ◽  
Vol 12 ◽  
Author(s):  
Giada Lo Gullo ◽  
Maria Luisa De Santis ◽  
Alessandro Paiardini ◽  
Serena Rosignoli ◽  
Alice Romagnoli ◽  
...  
Keyword(s):  
Large Subunit ◽  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Structural Data ◽  
Small Subunit ◽  
Large Ribosomal Subunit ◽  
Homologous Proteins ◽  
Gtp Hydrolysis ◽  
Elongation Factor 2 ◽  
Docking Protein

The translation factor IF6 is a protein of about 25 kDa shared by the Archaea and the Eukarya but absent in Bacteria. It acts as a ribosome anti-association factor that binds to the large subunit preventing the joining to the small subunit. It must be released from the large ribosomal subunit to permit its entry to the translation cycle. In Eukarya, this process occurs by the coordinated action of the GTPase Efl1 and the docking protein SBDS. Archaea do not possess a homolog of the former factor while they have a homolog of SBDS. In the past, we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homolog (aIF6) highlighting its similarity to the eukaryotic counterpart. Here, we analyzed the mechanism of aIF6 release from the large ribosomal subunit. We found that, similarly to the Eukarya, the detachment of aIF6 from the 50S subunit requires a GTPase activity which involves the archaeal elongation factor 2 (aEF-2). However, the release of aIF6 from the 50S subunits does not require the archaeal homolog of SBDS, being on the contrary inhibited by its presence. Molecular modeling, using published structural data of closely related homologous proteins, elucidated the mechanistic interplay between the aIF6, aSBDS, and aEF2 on the ribosome surface. The results suggest that a conformational rearrangement of aEF2, upon GTP hydrolysis, promotes aIF6 ejection. On the other hand, aSBDS and aEF2 share the same binding site, whose occupation by SBDS prevents aEF2 binding, thereby inhibiting aIF6 release.

Elongation factor Tu, a GTPase triggered by codon recognition on the ribosome: mechanism and GTP consumption

1995 ◽  
Vol 73 (11-12) ◽  
pp. 1221-1227 ◽  
Author(s):  
Marina V. Rodnina ◽  
Tillmann Pape ◽  
Rainer Fricke ◽  
Wolfgang Wintermeyer
Keyword(s):  
Ternary Complex ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Elongation Factor Tu ◽  
Gtp Hydrolysis ◽  
Time Resolved ◽  
Codon Recognition ◽  
Fluorescence Measurements ◽  
A Site

The mechanism of elongation factor Tu (EF-Tu) catalyzed aminoacyl-tRNA (aa-tRNA) binding to the A site of the ribosome was studied. Two types of complexes of EF-Tu with GTP and aa-tRNA, EF-Tu∙GTP∙aa-tRNA (ternary) and (EF-Tu∙GTP)2∙aa-tRNA (quinternary), can be formed in vitro depending on the conditions. On interaction with the ribosomal A site, generally only one molecule of GTP is hydrolysed per aa-tRNA bound and peptide bond formed. The second GTP molecule from the quinternary complex is hydrolyzed only during translation of an oligo(U) tract in the presence of EF-G. The first step in the interaction between the ribosome and the ternary complex is the codon-independent formation of an initial complex. In the absence of codon recognition, the aa-tRNA–EF-Tu complex does not enter further steps of A site binding and remains in the initial binding state. Despite the rapid formation of the initial complex, the rate constant of GTP hydrolysis in the noncognate complex is four orders of magnitude lower compared with the cognate complex. This, together with the results of time-resolved fluorescence measurements, suggests that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements that result in a conformational change of EF-Tu, presumably involving the effector region, which, in turn, triggers GTP hydrolysis and the subsequent steps of A site binding.Key words: translation, A site, codon recognition, fluorescence, stopped-flow.

scholarly journals Role of a ribosomal RNA phosphate oxygen during the EF-G–triggered GTP hydrolysis

2015 ◽  
Vol 112 (20) ◽  
pp. E2561-E2568 ◽  
Author(s):  
Miriam Koch ◽  
Sara Flür ◽  
Christoph Kreutz ◽  
Eric Ennifar ◽  
Ronald Micura ◽  
...  
Keyword(s):  
Ribosomal Rna ◽  
Electrostatic Interactions ◽  
Elongation Factor ◽  
Direct Interaction ◽  
Phosphate Group ◽  
Gtp Hydrolysis ◽  
Elongation Cycle ◽  
Close Proximity ◽  
Catalytically Active ◽  
Gtpase Activation

Elongation factor-catalyzed GTP hydrolysis is a key reaction during the ribosomal elongation cycle. Recent crystal structures of G proteins, such as elongation factor G (EF-G) bound to the ribosome, as well as many biochemical studies, provide evidence that the direct interaction of translational GTPases (trGTPases) with the sarcin-ricin loop (SRL) of ribosomal RNA (rRNA) is pivotal for hydrolysis. However, the precise mechanism remains elusive and is intensively debated. Based on the close proximity of the phosphate oxygen of A2662 of the SRL to the supposedly catalytic histidine of EF-G (His87), we probed this interaction by an atomic mutagenesis approach. We individually replaced either of the two nonbridging phosphate oxygens at A2662 with a methyl group by the introduction of a methylphosphonate instead of the natural phosphate in fully functional, reconstituted bacterial ribosomes. Our major finding was that only one of the two resulting diastereomers, the SP methylphosphonate, was compatible with efficient GTPase activation on EF-G. The same trend was observed for a second trGTPase, namely EF4 (LepA). In addition, we provide evidence that the negative charge of the A2662 phosphate group must be retained for uncompromised activity in GTP hydrolysis. In summary, our data strongly corroborate that the nonbridging proSP phosphate oxygen at the A2662 of the SRL is critically involved in the activation of GTP hydrolysis. A mechanistic scenario is supported in which positioning of the catalytically active, protonated His87 through electrostatic interactions with the A2662 phosphate group and H-bond networks are key features of ribosome-triggered activation of trGTPases.

scholarly journals Structural consequences of the interaction of RbgA with a 50S ribosomal subunit assembly intermediate

2019 ◽  
Vol 47 (19) ◽  
pp. 10414-10425 ◽  
Author(s):  
Amal Seffouh ◽  
Nikhil Jain ◽  
Dushyant Jahagirdar ◽  
Kaustuv Basu ◽  
Aida Razi ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Large Subunit ◽  
Ribosomal Subunit ◽  
Catalytic Residue ◽  
Gtp Hydrolysis ◽  
Subunit Assembly ◽  
Cryo Electron Microscopy ◽  
Assembly Factor ◽  
Assembly Intermediate ◽  
Immature Particle

Abstract Bacteria harbor a number GTPases that function in the assembly of the ribosome and are essential for growth. RbgA is one of these GTPases and is required for the assembly of the 50S subunit in most bacteria. Homologs of this protein are also implicated in the assembly of the large subunit of the mitochondrial and eukaryotic ribosome. We present here the cryo-electron microscopy structure of RbgA bound to a Bacillus subtilis 50S subunit assembly intermediate (45SRbgA particle) that accumulates in cells upon RbgA depletion. Binding of RbgA at the P site of the immature particle stabilizes functionally important rRNA helices in the A and P-sites, prior to the completion of the maturation process of the subunit. The structure also reveals the location of the highly conserved N-terminal end of RbgA containing the catalytic residue Histidine 9. The derived model supports a mechanism of GTP hydrolysis, and it shows that upon interaction of RbgA with the 45SRbgA particle, Histidine 9 positions itself near the nucleotide potentially acting as the catalytic residue with minimal rearrangements. This structure represents the first visualization of the conformational changes induced by an assembly factor in a bacterial subunit intermediate.

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