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 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 The mechanism of error induction by the antibiotic viomycin provides insight into the fidelity mechanism of translation

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Mikael Holm ◽  
Chandra Sekhar Mandava ◽  
Måns Ehrenberg ◽  
Suparna Sanyal
Keyword(s):  
Ternary Complex ◽  
Elongation Factor ◽  
Translation System ◽  
Elongation Factor Tu ◽  
Active Conformation ◽  
Cell Growth Inhibition ◽  
Gtp Hydrolysis ◽  
Steady State Kinetics ◽  
Initial Selection ◽  
Insight Into

Applying pre-steady state kinetics to an Escherichia-coli-based reconstituted translation system, we have studied how the antibiotic viomycin affects the accuracy of genetic code reading. We find that viomycin binds to translating ribosomes associated with a ternary complex (TC) consisting of elongation factor Tu (EF-Tu), aminoacyl tRNA and GTP, and locks the otherwise dynamically flipping monitoring bases A1492 and A1493 into their active conformation. This effectively prevents dissociation of near- and non-cognate TCs from the ribosome, thereby enhancing errors in initial selection. Moreover, viomycin shuts down proofreading-based error correction. Our results imply a mechanism in which the accuracy of initial selection is achieved by larger backward rate constants toward TC dissociation rather than by a smaller rate constant for GTP hydrolysis for near- and non-cognate TCs. Additionally, our results demonstrate that translocation inhibition, rather than error induction, is the major cause of cell growth inhibition by viomycin.

scholarly journals Two proofreading steps amplify the accuracy of genetic code translation

2016 ◽  
Vol 113 (48) ◽  
pp. 13744-13749 ◽  
Author(s):  
Ka-Weng Ieong ◽  
Ülkü Uzun ◽  
Maria Selmer ◽  
Måns Ehrenberg
Keyword(s):  
Protein Synthesis ◽  
Genetic Information ◽  
Molecular Basis ◽  
Messenger Rna ◽  
Ternary Complex ◽  
Elongation Factor ◽  
Elongation Factor Tu ◽  
Substrate Selection ◽  
Gtp Hydrolysis

Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aa-tRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EF-Tu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.

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 Oxazolidinone Antibiotics Target the P Site on Escherichiacoli Ribosomes

2002 ◽  
Vol 46 (4) ◽  
pp. 1080-1085 ◽  
Author(s):  
Hiroyuki Aoki ◽  
Lizhu Ke ◽  
Susan M. Poppe ◽  
Toni J. Poel ◽  
Elizabeth A. Weaver ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Wide Spectrum ◽  
Anaerobic Bacteria ◽  
Elongation Factor ◽  
Peptide Bond ◽  
23S Rrna ◽  
Peptide Bonds ◽  
A Site ◽  
Domain V ◽  
Trna Binding

ABSTRACT The oxazolidinones are a novel class of antimicrobial agents that target protein synthesis in a wide spectrum of gram-positive and anaerobic bacteria. The oxazolidinone PNU-100766 (linezolid) inhibits the binding of fMet-tRNA to 70S ribosomes. Mutations to oxazolidinone resistance in Halobacterium halobium, Staphylococcus aureus, and Escherichia coli map at or near domain V of the 23S rRNA, suggesting that the oxazolidinones may target the peptidyl transferase region responsible for binding fMet-tRNA. This study demonstrates that the potency of oxazolidinones corresponds to increased inhibition of fMet-tRNA binding. The inhibition of fMet-tRNA binding is competitive with respect to the fMet-tRNA concentration, suggesting that the P site is affected. The fMet-tRNA reacts with puromycin to form peptide bonds in the presence of elongation factor P (EF-P), which is needed for optimum specificity and efficiency of peptide bond synthesis. Oxazolidinone inhibition of the P site was evaluated by first binding fMet-tRNA to the A site, followed by translocation to the P site with EF-G. All three of the oxazolidinones used in this study inhibited translocation of fMet-tRNA. We propose that the oxazolidinones target the ribosomal P site and pleiotropically affect fMet-tRNA binding, EF-P stimulated synthesis of peptide bonds, and, most markedly, EF-G-mediated translocation of fMet-tRNA into the P site.

Dynamics and morphology of the in vitro polymeric form of elongation factor Tu from Escherichia coli

1996 ◽  
Vol 1291 (2) ◽  
pp. 122-130 ◽  
Author(s):  
Michael K. Helms ◽  
Gerard Marriott ◽  
William H. Sawyer ◽  
David M. Jameson
Keyword(s):  
Escherichia Coli ◽  
Elongation Factor ◽  
Elongation Factor Tu ◽  
Polymeric Form
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