Effect of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 on translocation

1. The effect of elongation factor 2 (EF 2) and of adenosine diphosphate-ribosylated elongation factor 2 (ADP-ribosyl-EF 2) on the shift of endogenous peptidyl-tRNA from the A to the P site of rat liver ribosomes (measured by the peptidyl-puromycin reaction) and on the release of deacylated tRNA (measured by aminoacylation) was investigated. 2. Limiting amounts of EF2, pre-bound or added to ribosomes, catalyse the shift of peptidyl-tRNA in the presence of GPT; when the enzyme is added in substrate amounts GMP-P(CH2)P [guanosine (beta, gamma-methylene)triphosphate] can partially replace GTP. ADP-ribosyl-EF 2 has no effect on the shift of peptidyl-tRNA when present in catalytic amounts, but becomes almost as effective as EF 2 when added in substrate amounts together with GTP; GMP-P(CH2)P cannot replace GTP. 3. The release of deacylated tRNA is induced only by substrate amounts of added EF 2 and also occurs in the absence of guanine nucleotides. In this reaction ADP-ribosyl-EF 2 is only 25% as effective as EF 2 in the absence of added nucleotide, but becomes 60-80% as effective in the presence of GTP or GMP-P(CH2)P. 4. The results obtained on protein-synthesizing systems are consistent with the hypothesis that ADP-ribosyl-EF 2 can operate a single round of translocation followed by binding of aminoacyl-tRNA and peptide-bond formation. 5. From the data obtained with the native enzyme it is concluded that the two moments of translocation require different conditions of interaction of EF 2 with ribosomes; it is suggested that the shift of peptidyl-tRNA is catalysed by EF 2 pre-bound to ribosomes, and that the release of tRNA is induced by a second molecule of interacting EF 2. The hydrolysis of GTP would be required for the release of pre-bound EF 2 from ribosomes. 5. The inhibition of the utilization of limiting amounts of EF 2 on ADP-ribosylation is very likely the consequence of a concomitant decrease in the rate of association and dissociation of the enzyme from ribosomes.

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Faculty Opinions recommendation of An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029397.349450 ◽

2005 ◽

Author(s):

Andrea Barta

Keyword(s):

Bond Formation ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Induced Fit ◽

Hydrolysis Of

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An induced-fit mechanism to promote peptide bond formation and exclude hydrolysis of peptidyl-tRNA

Structural Insights into Gene Expression and Protein Synthesis - Series in Structural Biology ◽

10.1142/9789811215865_0065 ◽

2020 ◽

pp. 552-556

Author(s):

T. Martin Schmeing ◽

Kevin S. Huang ◽

Scott A. Strobel ◽

Thomas A. Steitz

Keyword(s):

Bond Formation ◽

Peptide Bond ◽

Peptide Bond Formation ◽

Induced Fit ◽

Hydrolysis Of

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Irreversible chemical steps control intersubunit dynamics during translation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0805299105 ◽

2008 ◽

Vol 105 (40) ◽

pp. 15364-15369 ◽

Cited By ~ 82

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.

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

Science ◽

10.1126/science.1229017 ◽

2012 ◽

Vol 339 (6115) ◽

pp. 85-88 ◽

Cited By ~ 283

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.

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Elongation Factor P Interactions with the Ribosome Are Independent of Pausing

mBio ◽

10.1128/mbio.01056-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 2

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.

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Inhibition by ricin of protein synthesis in vitro. Inhibition of the binding of elongation factor 2 and of adenosine diphosphate-ribosylated elongation factor 2 to ribosomes

Biochemical Journal ◽

10.1042/bj1460127 ◽

1975 ◽

Vol 146 (1) ◽

pp. 127-131 ◽

Cited By ~ 99

Author(s):

L Montanaro ◽

S Sperti ◽

A Mattioli ◽

G Testoni ◽

F Stirpe

Keyword(s):

Protein Synthesis ◽

Binding Sites ◽

Polypeptide Chain ◽

Elongation Factor ◽

Adenosine Diphosphate ◽

Chain Elongation ◽

Elongation Factor 2 ◽

In Vitro Inhibition ◽

Liver Ribosomes

The binding of EF2 (elongation factor 2) and of ADP-ribosyl-EF 2 to rat liver ribosomes is inhibited by ricin. This result suggests that the native enzyme and its ADP-ribose derivative have the same or closely related binding sites on the ribosome. The inhibition by ricin of the binding of EF 2 to ribosomes is consistent with the previous observation that ricin affects EF 2-catalysed translocation during polypeptide chain elongation.

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Inhibition of protein synthesis in vitro by crotins and ricin. Effect on the steps of peptide chain elongation

Biochemical Journal ◽

10.1042/bj1560007 ◽

1976 ◽

Vol 156 (1) ◽

pp. 7-13 ◽

Cited By ~ 16

Author(s):

S Sperti ◽

L Montanaro ◽

A Mattioli ◽

G Testoni ◽

F Stirpe

Keyword(s):

Protein Synthesis ◽

Rat Liver ◽

Elongation Factor ◽

Artemia Salina ◽

Chain Elongation ◽

Gtp Hydrolysis ◽

Elongation Factor 2 ◽

The Individual ◽

Liver Ribosomes

The effects of crotin I and crotin II on the partial reactions of polypeptide chain elongation were investigated and compared with the known effects of ricin. Crotin II was a more powerful inhibitor than crotin I, but no qualitative differences between the two crotins were found. Rat liver ribosomes, preincubated with crotins and washed through sucrose gradients, remained inactive in protein synthesis. Among the individual steps of elongation, the peptidyltransferase reaction was unaffected by crotins, but some of the reactions that involve the interaction of elongation factors 1 and 2 with ribosomes were modified. A strong inhibition of the binding of elongation factor 2 to ribosomes and a stimulation of the elongation factor2-dependent GTP hydrolysis were observed; this indicates the formation of a very unstable elongation factor 2-GDP-ribosome complex, which, however, allows a single round of translocation to take place in the presence ofelongation factor 2 and added GTP. The elongation factor 1-dependent GTP hydrolysis was inhibited by crotins, whereas the enzymic binding of aminoacyl-tRNA, to both rat liver and Artemia salina ribosomes, was scarcely affected. In a protein-synthesizing system the inhibition by crotins and by ricin leads to a block of the nascent peptides on the ribosomal aminoacyl-tRNA site, an effect consistent with inhibition at the level of translocation. The mechanism of action of crotins appears to be very similar to that of ricin.

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Unwanted hydrolysis or α/β-peptide bond formation: how long should the rate-limiting coupling step take?

RSC Advances ◽

10.1039/c9ra06124j ◽

2019 ◽

Vol 9 (53) ◽

pp. 30720-30728 ◽

Cited By ~ 1

Author(s):

Viktória Goldschmidt Gőz ◽

Adrienn Nagy ◽

Viktor Farkas ◽

Ernő Keszei ◽

András Perczel

Keyword(s):

Amino Acids ◽

Bond Formation ◽

Side Reaction ◽

Peptide Bond ◽

Amide Bond ◽

Peptide Bond Formation ◽

Active Esters ◽

Amide Bond Formation ◽

Rate Limiting ◽

Hydrolysis Of

Parallel to the amide bond formation, the hydrolysis of the active esters of α/β-amino acids, as an unwanted side reaction limiting coupling efficacy, is studied.

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