SciCombinator

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Concept: Peptidyl transferase

175

The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the peptidyl transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the peptidyl transferase center.

Concepts: Protein, Bacteria, Amino acid, Ribosome, Messenger RNA, Protein biosynthesis, Chloramphenicol, Peptidyl transferase

5

The origins and evolution of the ribosome, 3-4 billion years ago, remain imprinted in the biochemistry of extant life and in the structure of the ribosome. Processes of ribosomal RNA (rRNA) expansion can be “observed” by comparing 3D rRNA structures of bacteria (small), yeast (medium), and metazoans (large). rRNA size correlates well with species complexity. Differences in ribosomes across species reveal that rRNA expansion segments have been added to rRNAs without perturbing the preexisting core. Here we show that rRNA growth occurs by a limited number of processes that include inserting a branch helix onto a preexisting trunk helix and elongation of a helix. rRNA expansions can leave distinctive atomic resolution fingerprints, which we call “insertion fingerprints.” Observation of insertion fingerprints in the ribosomal common core allows identification of probable ancestral expansion segments. Conceptually reversing these expansions allows extrapolation backward in time to generate models of primordial ribosomes. The approach presented here provides insight to the structure of pre-last universal common ancestor rRNAs and the subsequent expansions that shaped the peptidyl transferase center and the conserved core. We infer distinct phases of ribosomal evolution through which ribosomal particles evolve, acquiring coding and translocation, and extending and elaborating the exit tunnel.

Concepts: Protein, Gene, Archaea, Bacteria, RNA, Ribosomal RNA, Ribosome, Peptidyl transferase

4

In eukaryotes, accurate protein synthesis relies on a family of translational GTPases that pair with specific decoding factors to decipher the mRNA code on ribosomes. We present structures of the mammalian ribosome engaged with decoding factor⋅GTPase complexes representing intermediates of translation elongation (aminoacyl-tRNA⋅eEF1A), termination (eRF1⋅eRF3), and ribosome rescue (Pelota⋅Hbs1l). Comparative analyses reveal that each decoding factor exploits the plasticity of the ribosomal decoding center to differentially remodel ribosomal proteins and rRNA. This leads to varying degrees of large-scale ribosome movements and implies distinct mechanisms for communicating information from the decoding center to each GTPase. Additional structural snapshots of the translation termination pathway reveal the conformational changes that choreograph the accommodation of decoding factors into the peptidyl transferase center. Our results provide a structural framework for how different states of the mammalian ribosome are selectively recognized by the appropriate decoding factor⋅GTPase complex to ensure translational fidelity.

Concepts: Protein, Cell nucleus, RNA, Ribosome, Messenger RNA, Protein biosynthesis, Translation, Peptidyl transferase

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Accurate protein folding is essential for proper cellular and organismal function. In the cell, protein folding is carefully regulated; changes in folding homeostasis (proteostasis) can disrupt many cellular processes and have been implicated in various neurodegenerative diseases and other pathologies. For many proteins, the initial folding process begins during translation while the protein is still tethered to the ribosome; however, most biophysical studies of a protein’s energy landscape are carried out in isolation under idealized, dilute conditions and may not accurately report on the energy landscape in vivo. Thus, the energy landscape of ribosome nascent chains and the effect of the tethered ribosome on nascent chain folding remain unclear. Here we have developed a general assay for quantitatively measuring the folding stability of ribosome nascent chains, and find that the ribosome exerts a destabilizing effect on the polypeptide chain. This destabilization decreases as a function of the distance away from the peptidyl transferase center. Thus, the ribosome may add an additional layer of robustness to the protein-folding process by avoiding the formation of stable partially folded states before the protein has completely emerged from the ribosome.

Concepts: Protein, Protein structure, Gene, Amino acid, Metabolism, Ribosome, Protein folding, Peptidyl transferase

0

Several ATPases in the ATP-binding cassette F (ABCF) family confer resistance to macrolides, lincosamides and streptogramins (MLS) antibiotics. MLS are structurally distinct classes, but inhibit a common target: the peptidyl transferase (PTC) active site of the ribosome. Antibiotic resistance (ARE) ABCFs have recently been shown to operate through direct ribosomal protection, but the mechanistic details of this resistance mechanism are lacking. Using a reconstituted translational system, we dissect the molecular mechanism of Staphylococcus haemolyticus VgaALC and Enterococcus faecalis LsaA on the ribosome. We demonstrate that VgaALC is an NTPase that operates as a molecular machine strictly requiring NTP hydrolysis (not just NTP binding) for antibiotic protection. Moreover, when bound to the ribosome in the NTP-bound form, hydrolytically inactive EQ2 ABCF ARE mutants inhibit peptidyl transferase activity, suggesting a direct interaction between the ABCF ARE and the PTC. The likely structural candidate responsible for antibiotic displacement by wild type ABCF AREs, and PTC inhibition by the EQ2 mutant, is the extended inter-ABC domain linker region. Deletion of the linker region renders wild type VgaALC inactive in antibiotic protection and the EQ2 mutant inactive in PTC inhibition.

Concepts: Protein, Gene, Bacteria, Ribosome, Antibiotic resistance, Enterococcus, Enterococcus faecalis, Peptidyl transferase

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Antibiotic chloramphenicol (CHL) binds with a moderate affinity at the peptidyl transferase center of the bacterial ribosome and inhibits peptide bond formation. As an approach for modifying and potentially improving properties of this inhibitor, we explored ribosome binding and inhibitory activity of a number of amino-acid analogues of CHL. The L-histidyl analogue binds to the ribosome with the affinity exceeding that of CHL by 10 fold. Several of the newly synthesized analogues were able to inhibit protein synthesis and exhibited the mode of action that was distinct from the action of CHL. However, the inhibitory properties of the semi-synthetic CHL-analogues did not correlate with their affinity and in general, the amino-acid analogues of CHL were less active inhibitors of translation in comparison with the original antibiotic. The X-ray crystal structures of the Thermus thermophilus 70S ribosome in complex with three semi-synthetic analogues showed that CHL derivatives bind at the peptidyl transferase center, where the aminoacyl moiety of the tested compounds established idiosyncratic interactions with rRNA. Although still fairly inefficient inhibitors of translation, the synthesized compounds represent promising chemical scaffolds that target the peptidyl transferase center of the ribosome and potentially are suitable for further exploration.

Concepts: Protein, Bacteria, Amino acid, Ribosome, Peptide bond, Enzyme inhibitor, Inhibitor, Peptidyl transferase

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Understanding the mechanisms of inhibitors of translation termination may inform development of new antibacterials and therapeutics for premature-termination diseases. We report the crystal structure of the potent termination inhibitor BlaS bound to the ribosomal 70S•RF1 termination complex. BlaS shifts the catalytic domain 3 of RF1 and restructures the peptidyl transferase center. Universally conserved uridine 2585 in the peptidyl transferase center occludes the catalytic backbone of the GGQ motif of RF1, explaining the structural mechanism of inhibition. Rearrangement of domain 3 relative to the codon-recognition domain 2 provides insight into the dynamics of RF1 implicated in termination accuracy.

Concepts: Bacteria, Enzyme, Ribosome, Enzyme inhibitor, Solid, Inhibitor, Xanthine oxidase inhibitor, Peptidyl transferase

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A feasible scenario for the emergence of life requires the spontaneous materialization and sustainability of a proto-ribosome that could have catalyzed the formation of the first peptides. Models of proto-ribosomes were derived from the ribosomal PTC region, but the poor prebiotic copying abilities give rise to the question of their mode of replication. Here, complementarity is demonstrated in bacterial ribosomes, between nucleotides that constitute the two halves of the PTC cavity. The complementarity corroborates the dimeric nature of the proto-ribosome and is likely to underlie the symmetry of the PTC region. Furthermore, it indicates a simple and efficient replication mode; the strand of each monomer could have acted as a template for the synthesis of its counterpart, forming a self-replicating ribozyme. This article is protected by copyright. All rights reserved.

Concepts: Gene, Amino acid, RNA, Ribosome, Ribozyme, All rights reserved, Copyright, Peptidyl transferase

0

Bypassing is a recoding event that leads to the translation of two distal open reading frames into a single polypeptide chain. We present the structure of a translating ribosome stalled at the bypassing take-off site of gene 60 of bacteriophage T4. The nascent peptide in the exit tunnel anchors the P-site peptidyl-tRNA(Gly) to the ribosome and locks an inactive conformation of the peptidyl transferase center (PTC). The mRNA forms a short dynamic hairpin in the decoding site. The ribosomal subunits adopt a rolling conformation in which the rotation of the small subunit around its long axis causes the opening of the A-site region. Together, PTC conformation and mRNA structure safeguard against premature termination and read-through of the stop codon and reconfigure the ribosome to a state poised for take-off and sliding along the noncoding mRNA gap.

Concepts: DNA, Protein, Gene, Amino acid, Ribosome, Messenger RNA, Protein biosynthesis, Peptidyl transferase

0

McClary et al. (2017) identify the eukaryotic ribosome as a cellular target of agelastatin A, resolving the long-standing mystery surrounding the cytotoxic natural product’s mechanism of action. Structural and modeling studies further pinpointed the molecule’s binding site to the ribosome peptidyl transferase center, revealing key molecular interactions that drive binding.

Concepts: DNA, Electron, Ribosome, Matter, Atom, Atomic mass unit, Molecular modelling, Peptidyl transferase