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 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 Structural Characterization of an Alternative Mode of Tigecycline Binding to the Bacterial Ribosome

2015 ◽  
Vol 59 (5) ◽  
pp. 2849-2854 ◽  
Author(s):  
Andreas Schedlbauer ◽  
Tatsuya Kaminishi ◽  
Borja Ochoa-Lizarralde ◽  
Neha Dhimole ◽  
Shu Zhou ◽  
...  
Keyword(s):  
Ribosomal Subunit ◽  
Resistance Mechanisms ◽  
Side Chain ◽  
Alternative Mode ◽  
X Ray Crystallography ◽  
Bacterial Ribosome ◽  
A Site

ABSTRACTAlthough both tetracycline and tigecycline inhibit protein synthesis by sterically hindering the binding of tRNA to the ribosomal A site, tigecycline shows increased efficacy in bothin vitroandin vivoactivity assays and escapes the most common resistance mechanisms associated with the tetracycline class of antibiotics. These differences in activities are attributed to thetert-butyl-glycylamido side chain found in tigecycline. Our structural analysis by X-ray crystallography shows that tigecycline binds the bacterial 30S ribosomal subunit with its tail in an extended conformation and makes extensive interactions with the 16S rRNA nucleotide C1054. These interactions restrict the mobility of C1054 and contribute to the antimicrobial activity of tigecycline, including its resistance to the ribosomal protection proteins.

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 pH-sensitivity of the ribosomal peptidyl transfer reaction dependent on the identity of the A-site aminoacyl-tRNA

2010 ◽  
Vol 108 (1) ◽  
pp. 79-84 ◽  
Author(s):  
Magnus Johansson ◽  
Ka-Weng Ieong ◽  
Stefan Trobro ◽  
Peter Strazewski ◽  
Johan Åqvist ◽  
...  
Keyword(s):  
Ph Value ◽  
Ph Dependence ◽  
Peptide Bond ◽  
Bulk Solution ◽  
Peptide Bond Formation ◽  
Transfer Rates ◽  
Peptidyl Transfer ◽  
A Site ◽  
Rate Limiting ◽  
Site Binding

We studied the pH-dependence of ribosome catalyzed peptidyl transfer from fMet-tRNAfMet to the aa-tRNAs Phe-tRNAPhe, Ala-tRNAAla, Gly-tRNAGly, Pro-tRNAPro, Asn-tRNAAsn, and Ile-tRNAIle, selected to cover a large range of intrinsic pKa-values for the α-amino group of their amino acids. The peptidyl transfer rates were different at pH 7.5 and displayed different pH-dependence, quantified as the pH-value, , at which the rate was half maximal. The -values were downshifted relative to the intrinsic pKa-value of aa-tRNAs in bulk solution. Gly-tRNAGly had the smallest downshift, while Ile-tRNAIle and Ala-tRNAAla had the largest downshifts. These downshifts correlate strongly with molecular dynamics (MD) estimates of the downshifts in pKa-values of these aa-tRNAs upon A-site binding. Our data show the chemistry of peptide bond formation to be rate limiting for peptidyl transfer at pH 7.5 in the Gly and Pro cases and indicate rate limiting chemistry for all six aa-tRNAs.

scholarly journals A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines

2021 ◽  
Vol 12 ◽  
Author(s):  
Victor Barrenechea ◽  
Maryhory Vargas-Reyes ◽  
Miguel Quiliano ◽  
Pohl Milón
Keyword(s):  
Protein Synthesis ◽  
Translation Initiation ◽  
Ribosomal Subunit ◽  
Mrna Translation ◽  
Resistance Mechanisms ◽  
Dissociation Rate ◽  
Initiation Factor ◽  
Translation Elongation ◽  
Initiation Phase ◽  
Complementary Mechanism

Tetracycline has positively impacted human health as well as the farming and animal industries. Its extensive usage and versatility led to the spread of resistance mechanisms followed by the development of new variants of the antibiotic. Tetracyclines inhibit bacterial growth by impeding the binding of elongator tRNAs to the ribosome. However, a small number of reports indicated that Tetracyclines could also inhibit translation initiation, yet the molecular mechanism remained unknown. Here, we use biochemical and computational methods to study how Oxytetracycline (Otc), Demeclocycline (Dem), and Tigecycline (Tig) affect the translation initiation phase of protein synthesis. Our results show that all three Tetracyclines induce Initiation Factor IF3 to adopt a compact conformation on the 30S ribosomal subunit, similar to that induced by Initiation Factor IF1. This compaction was faster for Tig than Dem or Otc. Furthermore, all three tested tetracyclines affected IF1-bound 30S complexes. The dissociation rate constant of IF1 in early 30S complexes was 14-fold slower for Tig than Dem or Otc. Late 30S initiation complexes (30S pre-IC or IC) exhibited greater IF1 stabilization by Tig than for Dem and Otc. Tig and Otc delayed 50S joining to 30S initiation complexes (30S ICs). Remarkably, the presence of Tig considerably slowed the progression to translation elongation and retained IF1 in the resulting 70S initiation complex (70S IC). Molecular modeling of Tetracyclines bound to the 30S pre-IC and 30S IC indicated that the antibiotics binding site topography fluctuates along the initiation pathway. Mainly, 30S complexes show potential contacts between Dem or Tig with IF1, providing a structural rationale for the enhanced affinity of the antibiotics in the presence of the factor. Altogether, our data indicate that Tetracyclines inhibit translation initiation by allosterically perturbing the IF3 layout on the 30S, retaining IF1 during 70S IC formation, and slowing the transition toward translation elongation. Thus, this study describes a new complementary mechanism by which Tetracyclines may inhibit bacterial protein synthesis.

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 Large facilities and the evolving ribosome, the cellular machine for genetic-code translation

2009 ◽  
Vol 6 (suppl_5) ◽  
Author(s):  
Ada Yonath
Keyword(s):  
Synchrotron Radiation ◽  
Genetic Code ◽  
Structural Information ◽  
Peptide Bond ◽  
Polymerase Activity ◽  
Resistance Mechanisms ◽  
Peptide Bond Formation ◽  
Peptide Bonds ◽  
Polypeptide Chains

Well-focused X-ray beams, generated by advanced synchrotron radiation facilities, yielded high-resolution diffraction data from crystals of ribosomes, the cellular nano-machines that translate the genetic code into proteins. These structures revealed the decoding mechanism, localized the mRNA path and the positions of the tRNA molecules in the ribosome and illuminated the interactions of the ribosome with initiation, release and recycling factors. They also showed that the ribosome is a ribozyme whose active site is situated within a universal symmetrical region that is embedded in the otherwise asymmetric ribosome structure. As this highly conserved region provides the machinery required for peptide bond formation and for ribosome polymerase activity, it may be the remnant of the proto-ribosome, a dimeric pre-biotic machine that formed peptide bonds and non-coded polypeptide chains. Synchrotron radiation also enabled the determination of structures of complexes of ribosomes with antibiotics targeting them, which revealed the principles allowing for their clinical use, revealed resistance mechanisms and showed the bases for discriminating pathogens from hosts, hence providing valuable structural information for antibiotics improvement.

scholarly journals Structural evidence for a proline-specific glycopeptide recognition domain in an O-glycopeptidase

Glycobiology ◽  
2020 ◽  
Author(s):  
Ilit Noach ◽  
Alisdair B Boraston
Keyword(s):  
Active Sites ◽  
Substrate Recognition ◽  
Peptide Bond ◽  
Multidomain Protein ◽  
Threonine Residue ◽  
Tyrosine Residues ◽  
Host Pathogen Interaction ◽  
A Site ◽  
Insight Into

Abstract The glycosylation of proteins is typically considered as a stabilizing modification, including resistance to proteolysis. A class of peptidases, referred to as glycopeptidases or O-glycopeptidases, circumvent the protective effect of glycans against proteolysis by accommodating the glycans in their active sites as specific features of substrate recognition. IMPa from Pseudomonas aeruginosa is such an O-glycopeptidase that cleaves the peptide bond immediately preceding a site of O-glycosylation, and through this glycoprotein-degrading function contributes to the host-pathogen interaction. IMPa, however, is a relatively large multidomain protein and how its additional domains may contribute to its function remains unknown. Here, through the determination of a crystal structure of IMPa in complex with an O-glycopeptide, we reveal that the N-terminal domain of IMPa, which is classified in Pfam as IMPa_N_2, is a proline recognition domain that also shows the properties of recognizing an O-linked glycan on the serine/threonine residue following the proline. The proline is bound in the center of a bowl formed by four functionally conserved aromatic amino acid side chains while the glycan wraps around one of the tyrosine residues in the bowl to make classic aromatic ring-carbohydrate CH-π interactions. This structural evidence provides unprecedented insight into how the ancillary domains in glycoprotein-specific peptidases can noncatalytically recognize specific glycosylated motifs that are common in mucin and mucin-like molecules.

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.

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