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 Irreversible chemical steps control intersubunit dynamics during translation

2008 ◽  
Vol 105 (40) ◽  
pp. 15364-15369 ◽  
Author(s):  
R. Andrew Marshall ◽  
Magdalena Dorywalska ◽  
Joseph D. Puglisi
Keyword(s):  
Single Molecule ◽  
Conformational Changes ◽  
Large Scale ◽  
Bond Formation ◽  
Elongation Factor ◽  
Resonance Energy ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Gtp Hydrolysis ◽  
30S Subunit

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

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 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.

scholarly journals EF-P Is Essential for Rapid Synthesis of Proteins Containing Consecutive Proline Residues

Science ◽  
2012 ◽  
Vol 339 (6115) ◽  
pp. 85-88 ◽  
Author(s):  
Lili K. Doerfel ◽  
Ingo Wohlgemuth ◽  
Christina Kothe ◽  
Frank Peske ◽  
Henning Urlaub ◽  
...  
Keyword(s):  
Unknown Function ◽  
Bond Formation ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Transfer Rna ◽  
Peptide Bond Formation ◽  
Rapid Synthesis ◽  
Catalytic Center ◽  
Cellular Processes ◽  
Peptidyl Transfer

Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl–transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated β-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.

scholarly journals Elongation Factor P Interactions with the Ribosome Are Independent of Pausing

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Rodney Tollerson ◽  
Anne Witzky ◽  
Michael Ibba
Keyword(s):  
Single Molecule ◽  
Bond Formation ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Cellular Distribution ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
Single Molecule Tracking ◽  
Localization Pattern ◽  
Elongation Factor P

ABSTRACT Bacterial elongation factor P (EF-P) plays a pivotal role in the translation of polyproline motifs. To stimulate peptide bond formation, EF-P must enter the ribosome via an empty E-site. Using fluorescence-based single-molecule tracking, Mohapatra et al. (S. Mohapatra, H. Choi, X. Ge, S. Sanyal, and J. C. Weisshaar, mBio 8:e00300-17, 2017, https://doi.org/10.1128/mBio.00300-17 !) monitored the cellular distribution of EF-P and quantified the frequency of association between EF-P and the ribosome under various conditions. Findings from the study showed that EF-P has a localization pattern that is strikingly similar to that of ribosomes. Intriguingly, EF-P was seen to bind ribosomes more frequently than the estimated number of pausing events, indicating that E-site vacancies occur even when ribosomes are not paused. The study provides new insights into the mechanism of EF-P-dependent peptide bond formation and the intricacies of translation elongation.

scholarly journals The parable of the caveman and the Ferrari: protein synthesis and the RNA world

2017 ◽  
Vol 372 (1716) ◽  
pp. 20160187 ◽  
Author(s):  
Harry F. Noller
Keyword(s):  
Protein Synthesis ◽  
Ribosomal Rna ◽  
Rna World ◽  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Ribonucleoprotein Particle ◽  
Supportive Role

The basic steps of protein synthesis are carried out by the ribosome, a very large and complex ribonucleoprotein particle. In keeping with its proposed emergence from an RNA world, all three of its core mechanisms—aminoacyl-tRNA selection, catalysis of peptide bond formation and coupled translocation of mRNA and tRNA—are embodied in the properties of ribosomal RNA, while its proteins play a supportive role. This article is part of the themed issue ‘Perspectives on the ribosome’.

Ancient machinery embedded in the contemporary ribosome

2010 ◽  
Vol 38 (2) ◽  
pp. 422-427 ◽  
Author(s):  
Matthew J. Belousoff ◽  
Chen Davidovich ◽  
Ella Zimmerman ◽  
Yaron Caspi ◽  
Itai Wekselman ◽  
...  
Keyword(s):  
Gene Fusion ◽  
Bond Formation ◽  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Bacterial Organism ◽  
Or Gene ◽  
Definition Of ◽  
Folded Rna ◽  
Structural Definition

Structural analysis, supported by biochemical, mutagenesis and computational evidence, indicates that the peptidyltransferase centre of the contemporary ribosome is a universal symmetrical pocket composed solely of rRNA. This pocket seems to be a relic of the proto-ribosome, an ancient ribozyme, which was a dimeric RNA assembly formed from self-folded RNA chains of identical, similar or different sequences. This could have occurred spontaneously by gene duplication or gene fusion. This pocket-like entity was capable of autonomously catalysing various reactions, including peptide bond formation and non-coded or semi-coded amino acid polymerization. Efforts toward the structural definition of the early entity capable of genetic decoding involve the crystallization of the small ribosomal subunit of a bacterial organism harbouring a single functional rRNA operon.

scholarly journals Effect of modeccin on the steps of peptide-chain elongation

1978 ◽  
Vol 176 (2) ◽  
pp. 371-379 ◽  
Author(s):  
L Montanaro ◽  
S Sperti ◽  
M Zamboni ◽  
M Denaro ◽  
G Testoni ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Elongation Factor ◽  
Peptide Bond ◽  
Inhibitory Effect ◽  
Artemia Salina ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Experimental Conditions ◽  
Related Plant ◽  
The Individual

Modeccin inhibits polypeptide-chain elongation catalysed by Artemia salina (brine shrimp) ribosomes by inactivating the 60 S ribosomal subunit. Among the individual steps of elongation, peptide-bond formation, catalysed by 60 S peptidyltransferase, is unaffected by the toxin, whereas the binding of EF 2 (elongation factor 2) to ribosomes is strongly inhibited. Modeccin does not affect the poly(U)-dependent non-enzymic binding of either deacylated tRNAPhe or phenylalanyl-tRNA to ribosomes. The inhibitory effect of modeccin on the EF 1 (elongation factor 1)-dependent binding of phenylalanyl-tRNA is discussed, since it is decreased by tRNAPhe, which stimulates the binding reaction. The analysis of the distribution of ribosome-bound radioactivity during protein synthesis shows that modeccin consistently inhibits the radioactivity bound as long-chain peptides, but depending on the experimental conditions, can leave unchanged or even greatly stimulates the radioactivity bound as phenylalanyl-tRNA and/or short-chain peptides. It is concluded that, during the complete elongation cycle, modeccin does not affect the binding of the first aminoacyl-tRNA to ribosomes, but inhibits some step in the subsequent repetitive activity of either EF 1 or EF 2. The results obtained indicate that the mechanism of action of modeccin is very similar to that of ricin and related plant toxins such as abrin and crotin.

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