scholarly journals Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anne-Xander van der Stel ◽  
Emily R. Gordon ◽  
Arnab Sengupta ◽  
Allyson K. Martínez ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Structural Basis ◽  
Leader Peptide ◽  
Release Factor ◽  
Active Conformation ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Ribosome Stalling ◽  
Enhanced Sensitivity ◽  
Nascent Peptide ◽  
Recognition And Response

AbstractFree L-tryptophan (L-Trp) stalls ribosomes engaged in the synthesis of TnaC, a leader peptide controlling the expression of the Escherichia coli tryptophanase operon. Despite extensive characterization, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a TnaC variant (R23F) with greatly enhanced sensitivity for L-Trp. We show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. Importantly, the L-Trp binding site is not altered by the R23F mutation, suggesting that the relative rates of L-Trp binding and peptidyl-tRNA cleavage determine the tryptophan sensitivity of each variant. Thus, our study reveals a strategy whereby a nascent peptide assists the ribosome in detecting a small metabolite.

scholarly journals Structural basis for the tryptophan sensitivity of TnaC-mediated ribosome stalling

2021 ◽  
Author(s):  
Anne-Xander van der Stel ◽  
Emily R. Gordon ◽  
Arnab Sengupta ◽  
Allyson K. Martínez ◽  
Dorota Klepacki ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Structural Basis ◽  
Release Factor ◽  
Wild Type ◽  
Peptidyl Transferase ◽  
Ribosome Stalling ◽  
Bacterial Ribosome ◽  
Translational Arrest ◽  
Operon Expression ◽  
Recognition And Response

ABSTRACTFree L-tryptophan (L-Trp) induces the expression of the Escherichia coli tryptophanase operon, leading to the production of indole from L-Trp. Tryptophanase operon expression is controlled via a mechanism involving the tryptophan-dependent stalling of ribosomes engaged in translation of tnaC, a leader sequence upstream of tnaA that encodes a 24-residue peptide functioning as a sensor for L-Trp. Although extensive biochemical characterization has revealed the elements of the TnaC peptide and the ribosome that are responsible for translational arrest, the molecular mechanism underlying the recognition and response to L-Trp by the TnaC-ribosome complex remains unknown. Here, we use a combined biochemical and structural approach to characterize a variant of TnaC (R23F) in which stalling by L-Trp is enhanced because of reduced cleavage of TnaC(R23F)-peptidyl-tRNA. In contrast to previous data originated from lower resolution structural studies, we show that the TnaC–ribosome complex captures a single L-Trp molecule to undergo tryptophan-dependent termination arrest and that nascent TnaC prevents the catalytic GGQ loop of release factor 2 from adopting an active conformation at the peptidyl transferase center. In addition, we show that the conformation of the L-Trp binding site is not altered by the R23F mutation. This leads us to propose a model in which rates of TnaC-peptidyl-tRNA cleavage by release factor and binding of the L-Trp ligand to the translating ribosome determine the tryptophan sensitivity of the wild-type and mutant TnaC variants. Thus, our study reveals a strategy whereby a nascent peptide assists the bacterial ribosome in sensing a small metabolite.

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 Mechanism of ribosome rescue by ArfA and RF2

2016 ◽  
Author(s):  
Gabriel Demo ◽  
Egor Svidritskiy ◽  
Rohini Madireddy ◽  
Ruben Diaz-Avalos ◽  
Timothy Grant ◽  
...  
Keyword(s):  
Structural Dynamics ◽  
Stop Codon ◽  
Release Factor ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
C Terminus ◽  
Peptide Release ◽  
N Terminus ◽  
70S Ribosome ◽  
A Site

AbstractArfA rescues ribosomes stalled on truncated mRNAs by recruiting the release factor RF2, which normally binds stop codons to catalyze peptide release. We report two 3.2-Å resolution cryo-EM structures – determined from a single sample – of the 70S ribosome with ArfA∙RF2 in the A site. In both states, the ArfA C-terminus occupies the mRNA tunnel downstream of the A site. One state contains a compact inactive RF2 conformation, hitherto unobserved in 70S termination complexes. Ordering of the ArfA N-terminus in the second state rearranges RF2 into an extended conformation that docks the catalytic GGQ motif into the peptidyl-transferase center. Our work thus reveals the structural dynamics of ribosome rescue. The structures demonstrate how ArfA “senses” the vacant mRNA tunnel and activates RF2 to mediate peptide release without a stop codon, allowing stalled ribosomes to be recycled.

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 Ornithine capture by a translating ribosome controls bacterial polyamine synthesis

2019 ◽  
Author(s):  
Alba Herrero del Valle ◽  
Britta Seip ◽  
Iñaki Cervera-Marzal ◽  
Guénaël Sacheau ◽  
A. Carolin Seefeldt ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Binding Pocket ◽  
Growth Stress ◽  
Release Factor ◽  
Human Pathogens ◽  
Polyamine Synthesis ◽  
E Coli ◽  
Ribosome Stalling ◽  
Nascent Peptide ◽  
Transcription Antitermination

ABSTRACTPolyamines are essential metabolites that play an important role in cell growth, stress adaptation, and microbial virulence1–3. In order to survive and multiply within a human host, pathogenic bacteria adjust the expression and activity of polyamine biosynthetic enzymes in response to different environmental stresses and metabolic cues2. Here, we show that ornithine capture by the ribosome and the nascent peptide SpeFL controls polyamine synthesis in γ-proteobacteria by inducing the expression of the ornithine decarboxylase SpeF4, via a mechanism involving ribosome stalling and transcription antitermination. In addition, we present the cryo-EM structure of an Escherichia coli (E. coli) ribosome stalled during translation of speFL in the presence of ornithine. The structure shows how the ribosome and the SpeFL sensor domain form a highly selective binding pocket that accommodates a single ornithine molecule but excludes near-cognate ligands. Ornithine pre-associates with the ribosome and is then held in place by the sensor domain, leading to the compaction of the SpeFL effector domain and blocking the action of release factor RF1. Thus, our study not only reveals basic strategies by which nascent peptides assist the ribosome in detecting a specific metabolite, but also provides a framework for assessing how ornithine promotes virulence in several human pathogens.

scholarly journals Structure-Activity Relationships of Diverse Oxazolidinones for Linezolid-Resistant Staphylococcus aureus Strains Possessing the cfr Methyltransferase Gene or Ribosomal Mutations

2010 ◽  
Vol 54 (12) ◽  
pp. 5337-5343 ◽  
Author(s):  
Jeffrey B. Locke ◽  
John Finn ◽  
Mark Hilgers ◽  
Gracia Morales ◽  
Shahad Rahawi ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ribosomal Proteins ◽  
Resistance Mechanisms ◽  
Structural Basis ◽  
23S Rrna ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Structure Activity ◽  
Methyltransferase Gene ◽  
Key Features

ABSTRACT Staphylococcal resistance to linezolid (LZD) is mediated through ribosomal mutations (23S rRNA or ribosomal proteins L3 and L4) or through methylation of 23S rRNA by the horizontally transferred Cfr methyltransferase. To investigate the structural basis for oxazolidinone activity against LZD-resistant (LZDr) strains, we compared structurally diverse, clinically relevant oxazolidinones, including LZD, radezolid (RX-1741), TR-700 (torezolid), and a set of TR-700 analogs (including novel CD-rings and various A-ring C-5 substituents), against a panel of laboratory-derived and clinical LZDr Staphylococcus aureus strains possessing a variety of resistance mechanisms. Potency against all strains was correlated with optimization of C- and D-rings, which interact with more highly conserved regions of the peptidyl transferase center binding site. Activity against cfr strains was retained with either hydroxymethyl or 1,2,3-triazole C-5 groups but was reduced by 2- to 8-fold in compounds with acetamide substituents. LZD, which possesses a C-5 acetamide group and lacks a D-ring substituent, demonstrated the lowest potency against all strains tested, particularly against cfr strains. These data reveal key features contributing to oxazolidinone activity and highlight structural tradeoffs between potency against susceptible strains and potency against strains with various resistance mechanisms.

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
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