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.

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

1976 ◽  
Vol 156 (1) ◽  
pp. 7-13 ◽  
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.

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.

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 Orthoformimycin inhibits translation elongation by displacing the A-site tRNA and preventing peptide bond formation

2021 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Tatsuya Kaminishi ◽  
Attilio Fabbretti ◽  
Pohl Milon ◽  
Xu Han ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Peptide Bond ◽  
Resistance Mechanisms ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
X Ray Crystallography ◽  
Steady State Kinetics ◽  
A Site ◽  
Insight Into ◽  
Major Target

The ribosome is a major target for antibiotics owing to its essential cellular role in protein synthesis. Structural analysis of ribosome-antibiotic complexes provides insight into the molecular basis for their inhibitory action and highlights possible avenues to improve their potential or overcome existing resistance mechanisms. Here we use X-ray crystallography and pre-steady state kinetics to detail the inhibitory mechanism of the antimicrobial on the large ribosomal subunit.

scholarly journals Alkaloid homoharringtonine inhibits polypeptide chain elongation on human ribosomes on the step of peptide bond formation

FEBS Letters ◽  
1989 ◽  
Vol 257 (2) ◽  
pp. 254-256 ◽  
Author(s):  
R.M. Tujebajeva ◽  
D.M. Graifer ◽  
G.G. Karpova ◽  
N.A. Ajtkhozhina
Keyword(s):  
Polypeptide Chain ◽  
Bond Formation ◽  
Peptide Bond ◽  
Chain Elongation ◽  
Peptide Bond Formation

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 Ribosome crystallography: catalysis and evolution of peptide-bond formation, nascent chain elongation and its co-translational folding

2005 ◽  
Vol 33 (3) ◽  
pp. 488-492 ◽  
Author(s):  
A. Bashan ◽  
A. Yonath
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Trigger Factor ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Chemical Catalysis ◽  
Nascent Chain ◽  
Peptidyl Transferase Centre ◽  
Folding Space

A ribosome is a ribozyme polymerizing amino acids, exploiting positional- and substrate-mediated chemical catalysis. We showed that peptide-bond formation is facilitated by the ribosomal architectural frame, provided by a sizable symmetry-related region in and around the peptidyl transferase centre, suggesting that the ribosomal active site was evolved by gene fusion. Mobility in tunnel components is exploited for elongation arrest as well as for trafficking nascent proteins into the folding space bordered by the bacterial chaperone, namely the trigger factor.

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