scholarly journals Disome-seq reveals widespread ribosome collisions that recruit co-translational chaperones

2019 ◽  
Author(s):  
Taolan Zhao ◽  
Yan-Ming Chen ◽  
Yu Li ◽  
Jia Wang ◽  
Siyu Chen ◽  
...  
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Protein Homeostasis ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
Protein Levels ◽  
Cryo Electron Microscopy ◽  
Exit Tunnel ◽  
Hidden Layer ◽  
A Site

ABSTRACTRegulation of translation elongation plays a crucial role in determining absolute protein levels and ensuring the correct localization and folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes (ribo-seq). However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes — a product of translational pauses during which the 5′-ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are missed in traditional ribo-seq. These collisions are due to 1) slow ribosome release when stop codons are at the A-site, 2) slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and 3) slow leaving of polylysine from the exit tunnel of ribosomes. The paused ribosomes can continue translating after collisions, as suggested by the structure of disomes obtained by cryo-electron microscopy (cryo-EM). Collided ribosomes recruit chaperones, which can aid in the co-translational folding of the nascent peptides. Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

scholarly journals Disome-seq reveals widespread ribosome collisions that promote cotranslational protein folding

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Taolan Zhao ◽  
Yan-Ming Chen ◽  
Yu Li ◽  
Jia Wang ◽  
Siyu Chen ◽  
...  
Keyword(s):  
Protein Quality ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Translation Elongation ◽  
Cryo Electron Microscopy ◽  
Cotranslational Folding ◽  
A Cell ◽  
Exit Tunnel ◽  
Hidden Layer ◽  
A Site

Abstract Background The folding of proteins is challenging in the highly crowded and sticky environment of a cell. Regulation of translation elongation may play a crucial role in ensuring the correct folding of proteins. Much of our knowledge regarding translation elongation comes from the sequencing of mRNA fragments protected by single ribosomes by ribo-seq. However, larger protected mRNA fragments have been observed, suggesting the existence of an alternative and previously hidden layer of regulation. Results In this study, we performed disome-seq to sequence mRNA fragments protected by two stacked ribosomes, a product of translational pauses during which the 5′-elongating ribosome collides with the 3′-paused one. We detected widespread ribosome collisions that are related to slow ribosome release when stop codons are at the A-site, slow peptide bond formation from proline, glycine, asparagine, and cysteine when they are at the P-site, and slow leaving of polylysine from the exit tunnel of ribosomes. The structure of disomes obtained by cryo-electron microscopy suggests a different conformation from the substrate of the ribosome-associated protein quality control pathway. Collisions occurred more frequently in the gap regions between α-helices, where a translational pause can prevent the folding interference from the downstream peptides. Paused or collided ribosomes are associated with specific chaperones, which can aid in the cotranslational folding of the nascent peptides. Conclusions Therefore, cells use regulated ribosome collisions to ensure protein homeostasis.

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.

ONIOM and ab-initio calculations on the mechanism of uncatalyzed peptide bond formation

2012 ◽  
Vol 90 (6) ◽  
pp. 691-700 ◽  
Author(s):  
Hadieh Monajemi ◽  
Mohammad Noh Daud ◽  
Sharifuddin Mohd. Zain ◽  
Wan Ahmad Tajuddin Wan Abdullah
Keyword(s):  
Amino Acids ◽  
Bond Formation ◽  
Computational Cost ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Initiator Trna ◽  
Reduced Rate ◽  
Surface Scan ◽  
A Site

Finding a proper transition structure for the peptide bond formation process can lead one to a better understanding of the role of ribosome in catalyzing this reaction. Using computer simulations, we performed the potential energy surface scan on the ester bond dissociation of P-site aminoacyl-tRNA and the peptide bond formation of P-site and A-site amino acids. The full fragments of initiator tRNAimet and elongator tRNAphe are attached to both cognate and non-cognate amino acids as the P-site substrate. The A-site amino acid for all four calculations is methionine. We used ONIOM calculations to reduce the computational cost. Our study illustrates the reduced rate of peptide bond formation for misacylated tRNAimet in the absence of ribosomal bases. The misacylated elongator tRNAphe, however, did not show any difference in its PES compared with that for the phe-tRNAphe. This demonstrates the structural specification of initiator tRNAimet for the amino acids side chain.

scholarly journals Visualization of Trna Movements on the Escherichia coli 70s Ribosome during the Elongation Cycle

2000 ◽  
Vol 150 (3) ◽  
pp. 447-460 ◽  
Author(s):  
Rajendra K. Agrawal ◽  
Christian M.T. Spahn ◽  
Pawel Penczek ◽  
Robert A. Grassucci ◽  
Knud H. Nierhaus ◽  
...  
Keyword(s):  
Bond Formation ◽  
Three Dimensional ◽  
Peptide Bond ◽  
High Accuracy ◽  
Peptide Bond Formation ◽  
Elongation Cycle ◽  
Before And After ◽  
70S Ribosome ◽  
Similar Accuracy ◽  
A Site

Three-dimensional cryomaps have been reconstructed for tRNA–ribosome complexes in pre- and posttranslocational states at 17-Å resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNAfMet-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon–anticodon interaction at the E site appears feasible.

scholarly journals Peptidyl transferase center decompaction and structural constraints during early protein elongation on the ribosome

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Bin Jia ◽  
Tianlong Wang ◽  
Jean Lehmann
Keyword(s):  
Peptide Bond ◽  
Nucleophilic Attack ◽  
Structural Constraints ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Early Protein ◽  
Physico Chemical ◽  
Exit Tunnel ◽  
A Site

AbstractPeptide bond formation on the ribosome requires that aminoacyl-tRNAs and peptidyl-tRNAs are properly positioned on the A site and the P site of the peptidyl transferase center (PTC) so that nucleophilic attack can occur. Here we analyse some constraints associated with the induced-fit mechanism of the PTC, that promotes this positioning through a compaction around the aminoacyl ester orchestrated by U2506. The physical basis of PTC decompaction, that allows the elongated peptidyl-tRNA to free itself from that state and move to the P site of the PTC, is still unclear. From thermodynamics considerations and an analysis of published ribosome structures, the present work highlights the rational of this mechanism, in which the free-energy released by the new peptide bond is used to kick U2506 away from the reaction center. Furthermore, we show the evidence that decompaction is impaired when the nascent peptide is not yet anchored inside the exit tunnel, which may contribute to explain why the first rounds of elongation are inefficient, an issue that has attracted much interest for about two decades. Results in this field are examined in the light of the present analysis and a physico-chemical correlation in the genetic code, which suggest that elementary constraints associated with the size of the side-chain of the amino acids penalize early elongation events.

scholarly journals Insights into the base-pairing preferences of 8-oxoguanosine on the ribosome

2019 ◽  
Vol 47 (18) ◽  
pp. 9857-9870 ◽  
Author(s):  
Erica N Thomas ◽  
Carrie L Simms ◽  
Hannah E Keedy ◽  
Hani S Zaher
Keyword(s):  
Base Pair ◽  
Structural Changes ◽  
Bond Formation ◽  
Peptide Bond ◽  
Underlying Mechanism ◽  
Peptide Bond Formation ◽  
Base Pairing ◽  
Base Pairs ◽  
Free Dna ◽  
A Site

Abstract Of the four bases, guanine is the most susceptible to oxidation, which results in the formation of 8-oxoguanine (8-oxoG). In protein-free DNA, 8-oxodG adopts the syn conformation more frequently than the anti one. In the syn conformation, 8-oxodG base pairs with dA. The equilibrium between the anti and syn conformations of the adduct are known to be altered by the enzyme recognizing 8-oxodG. We previously showed that 8-oxoG in mRNA severely disrupts tRNA selection, but the underlying mechanism for these effects was not addressed. Here, we use miscoding antibiotics and ribosome mutants to probe how 8-oxoG interacts with the tRNA anticodon in the decoding center. Addition of antibiotics and introduction of error-inducing mutations partially suppressed the effects of 8-oxoG. Under these conditions, rates and/or endpoints of peptide-bond formation for the cognate (8-oxoG•C) and near-cognate (8-oxoG•A) aminoacyl-tRNAs increased. In contrast, the antibiotics had little effect on other mismatches, suggesting that the lesion restricts the nucleotide from forming other interactions. Our findings suggest that 8-oxoG predominantly adopts the syn conformation in the A site. However, its ability to base pair with adenosine in this conformation is not sufficient to promote the necessary structural changes for tRNA selection to proceed.

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