scholarly journals Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

2018 ◽  
Author(s):  
Wenfei Li ◽  
Fred R. Ward ◽  
Kim F. McClure ◽  
Stacey Tsai-Lan Chang ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Structural Basis ◽  
Small Subset ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractSmall molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3’-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

scholarly journals Selective inhibition of human translation termination by a drug-like compound

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wenfei Li ◽  
Stacey Tsai-Lan Chang ◽  
Fred. R. Ward ◽  
Jamie H. D. Cate
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Selective Inhibition ◽  
Helical Conformation ◽  
Eukaryotic Translation ◽  
Inhibit Gene Expression ◽  
Nascent Chain ◽  
Specific Proteins ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Abstract Methods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation. These molecules also inhibit translation termination by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 inhibits translation termination by arresting the nascent chain (NC) in the ribosome exit tunnel. The arrested NC adopts a compact α-helical conformation that induces 28 S rRNA nucleotide rearrangements that suppress the peptidyl transferase center (PTC) catalytic activity stimulated by eukaryotic release factor 1 (eRF1). These data support a mechanism of action for a small molecule targeting translation that suppresses peptidyl-tRNA hydrolysis promoted by eRF1, revealing principles of eukaryotic translation termination and laying the foundation for new therapeutic strategies.

scholarly journals Selective inhibition of human translation by a drug-like compound that traps terminated protein nascent chains on the ribosome

2020 ◽  
Author(s):  
Wenfei Li ◽  
Stacey Tsai-Lan Chang ◽  
Fred. R Ward ◽  
Jamie H. D. Cate
Keyword(s):  
Small Molecules ◽  
Stop Codon ◽  
Translation Termination ◽  
Selective Inhibition ◽  
Helical Conformation ◽  
28S Rrna ◽  
Inhibit Gene Expression ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractMethods to directly inhibit gene expression using small molecules hold promise for the development of new therapeutics targeting proteins that have evaded previous attempts at drug discovery. Among these, small molecules including the drug-like compound PF-06446846 (PF846) selectively inhibit the synthesis of specific proteins, by stalling translation elongation 1–4. These molecules also inhibit translation termination 4 by an unknown mechanism. Using cryo-electron microscopy (cryo-EM) and biochemical approaches, we show that PF846 arrests translation at the stop codon by slowing hydrolysis of the protein nascent chain (NC) from peptidyl-site (P-site) tRNA by eukaryotic release factor 1 (eRF1). After NC hydrolysis from the P-site tRNA, PF846 traps the NC in the ribosome exit tunnel in a compact α-helical conformation that induces 28S rRNA nucleotide rearrangements propagating back to the ribosome peptidyl transferase center (PTC). Mutational analyses and human cell-based experiments elucidate the pivotal amino acids of the NC required for PF846-dependent termination arrest, all of which face the PF846 side of the ribosome exit tunnel. The structural and functional data support a model in which PF846 inhibits translation termination by inducing allosteric conformational rearrangements in the NC and PTC that suppress peptidyl-tRNA hydrolysis promoted by eRF1, and trap the NC in the ribosome exit tunnel. This unprecedented mechanism of action reveals new principles of translation termination and lays the foundation for new therapeutic strategies.

scholarly journals Folding of VemP into translation-arresting secondary structure is driven by the ribosome exit tunnel

2021 ◽  
Author(s):  
Michal H Kolar ◽  
Gabor Nagy ◽  
John Kunkel ◽  
Sara M Vaiana ◽  
Lars V Bock ◽  
...  
Keyword(s):  
Small Molecules ◽  
Protein Production ◽  
Driving Forces ◽  
Peptidyl Transferase ◽  
Helix Formation ◽  
Translational Arrest ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
Α Helix ◽  
Dynamics Simulations

The ribosome is a fundamental biomolecular complex responsible for protein production in cells. Nascent proteins emerge from the ribosome through a tunnel, where they may interact with the tunnel walls or small molecules such as antibiotics. These interactions can cause translational arrest with notable physiologic consequences. Here, we studied the arrest caused by the regulatory peptide VemP, which is known to form an α-helix in the ribosome tunnel near the peptidyl transferase center under specific conditions. We used all-atom molecular dynamics simulations of the entire ribosome and circular dichroism spectroscopy to study the driving forces of helix formation and how VemP causes the translational arrest. To that aim, we compared VemP dynamics in the ribosome tunnel with its dynamics in solution. We show that the VemP sequence has a low helical propensity in water and that the propensity is higher in more hydrophobic solvents. We propose that helix formation within the ribosome is driven by the tunnel environment and that a portion of VemP acts as an anchor. This anchor might slow down VemP progression through the tunnel enabling the α-helix formation, which causes the elongation arrest.

scholarly journals Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

2019 ◽  
Author(s):  
Abid Javed ◽  
Tomasz Wlodarski ◽  
Anaïs. M.E. Cassaignau ◽  
Lisa D. Cabrita ◽  
John Christodoulou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Active Component ◽  
Chain Dynamics ◽  
Cryo Electron Microscopy ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Immunoglobulin Domain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

scholarly journals Nascent Peptide in the Ribosome Exit Tunnel Affects Functional Properties of the A-Site of the Peptidyl Transferase Center

2011 ◽  
Vol 41 (3) ◽  
pp. 321-330 ◽  
Author(s):  
Haripriya Ramu ◽  
Nora Vázquez-Laslop ◽  
Dorota Klepacki ◽  
Qing Dai ◽  
Joseph Piccirilli ◽  
...  
Keyword(s):  
Functional Properties ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Nascent Peptide ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel ◽  
A Site

scholarly journals Ribosomal Features Essential for tna Operon Induction: Tryptophan Binding at the Peptidyl Transferase Center

2007 ◽  
Vol 189 (8) ◽  
pp. 3140-3146 ◽  
Author(s):  
Luis R. Cruz-Vera ◽  
Aaron New ◽  
Catherine Squires ◽  
Charles Yanofsky
Keyword(s):  
23S Rrna ◽  
Transferase Activity ◽  
Wild Type ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Free Tryptophan ◽  
Ribosomal Protein L22 ◽  
Exit Tunnel ◽  
Operon Expression ◽  
Ribosome Exit Tunnel

ABSTRACT Features of the amino acid sequence of the TnaC nascent peptide are recognized by the translating ribosome. Recognition leads to tryptophan binding within the translating ribosome, inhibiting the termination of tnaC translation and preventing Rho-dependent transcription termination in the tna operon leader region. It was previously shown that inserting an adenine residue at position 751 or introducing the U2609C change in 23S rRNA or introducing the K90W replacement in ribosomal protein L22 prevented tryptophan induction of tna operon expression. It was also observed that an adenine at position 752 of 23S rRNA was required for induction. In the current study, the explanation for the lack of induction by these altered ribosomes was investigated. Using isolated TnaC-ribosome complexes, it was shown that although tryptophan inhibits puromycin cleavage of TnaC-tRNAPro with wild-type ribosome complexes, it does not inhibit cleavage with the four mutant ribosome complexes examined. Similarly, tryptophan prevents sparsomycin inhibition of TnaC-tRNAPro cleavage with wild-type ribosome complexes but not with these mutant ribosome complexes. Additionally, a nucleotide located close to the peptidyl transferase center, A2572, which was protected from methylation by tryptophan with wild-type ribosome complexes, was not protected with mutant ribosome complexes. These findings identify specific ribosomal residues located in the ribosome exit tunnel that recognize features of the TnaC peptide. This recognition creates a free tryptophan-binding site in the peptidyl transferase center, where bound tryptophan inhibits peptidyl transferase activity.

scholarly journals The GTPase Nog1 co-ordinates the assembly, maturation and quality control of distant ribosomal functional centers

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Purnima Klingauf-Nerurkar ◽  
Ludovic C Gillet ◽  
Daniela Portugal-Calisto ◽  
Michaela Oborská-Oplová ◽  
Martin Jäger ◽  
...  
Keyword(s):  
Quality Control ◽  
Protein Folding ◽  
Atpase Activity ◽  
Ribosomal Protein ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Eukaryotic Ribosome ◽  
Ribosomal Stalk ◽  
Exit Tunnel ◽  
Ribosome Formation

Eukaryotic ribosome precursors acquire translation competence in the cytoplasm through stepwise release of bound assembly factors, and proofreading of their functional centers. In case of the pre-60S, these steps include removal of placeholders Rlp24, Arx1 and Mrt4 that prevent premature loading of the ribosomal protein eL24, the protein-folding machinery at the polypeptide exit tunnel (PET), and the ribosomal stalk, respectively. Here, we reveal that sequential ATPase and GTPase activities license release factors Rei1 and Yvh1 to trigger Arx1 and Mrt4 removal. Drg1-ATPase activity removes Rlp24 from the GTPase Nog1 on the pre-60S; consequently, the C-terminal tail of Nog1 is extracted from the PET. These events enable Rei1 to probe PET integrity and catalyze Arx1 release. Concomitantly, Nog1 eviction from the pre-60S permits peptidyl transferase center maturation, and allows Yvh1 to mediate Mrt4 release for stalk assembly. Thus, Nog1 co-ordinates the assembly, maturation and quality control of distant functional centers during ribosome formation.

scholarly journals Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

2020 ◽  
Author(s):  
Anna B. Loveland ◽  
Egor Svidritskiy ◽  
Denis Susorov ◽  
Soojin Lee ◽  
Alexander Park ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Specific Binding ◽  
Structural Basis ◽  
Transferase Activity ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Ribosomal Tunnel ◽  
Cellular Translation ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

AbstractToxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

scholarly journals Ribosome protection by antibiotic resistance ATP-binding cassette protein

2018 ◽  
Vol 115 (20) ◽  
pp. 5157-5162 ◽  
Author(s):  
Weixin Su ◽  
Veerendra Kumar ◽  
Yichen Ding ◽  
Rya Ero ◽  
Aida Serra ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Conformational Changes ◽  
Atp Binding ◽  
Peptidyl Transferase ◽  
Exit Site ◽  
Peptidyl Transferase Center ◽  
Biochemical Assays ◽  
Exit Tunnel ◽  
Insight Into ◽  
Atp Binding Cassette

The ribosome is one of the richest targets for antibiotics. Unfortunately, antibiotic resistance is an urgent issue in clinical practice. Several ATP-binding cassette family proteins confer resistance to ribosome-targeting antibiotics through a yet unknown mechanism. Among them, MsrE has been implicated in macrolide resistance. Here, we report the cryo-EM structure of ATP form MsrE bound to the ribosome. Unlike previously characterized ribosomal protection proteins, MsrE is shown to bind to ribosomal exit site. Our structure reveals that the domain linker forms a unique needle-like arrangement with two crossed helices connected by an extended loop projecting into the peptidyl-transferase center and the nascent peptide exit tunnel, where numerous antibiotics bind. In combination with biochemical assays, our structure provides insight into how MsrE binding leads to conformational changes, which results in the release of the drug. This mechanism appears to be universal for the ABC-F type ribosome protection proteins.

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