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

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.

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

10.1101/315325 ◽

2018 ◽

Cited By ~ 3

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.

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

Journal of Bacteriology ◽

10.1128/jb.01869-06 ◽

2007 ◽

Vol 189 (8) ◽

pp. 3140-3146 ◽

Cited By ~ 31

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.

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

eLife ◽

10.7554/elife.52474 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 7

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.

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Ribosome protection by antibiotic resistance ATP-binding cassette protein

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1803313115 ◽

2018 ◽

Vol 115 (20) ◽

pp. 5157-5162 ◽

Cited By ~ 26

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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Mechanism of ribosome rescue by ArfA and RF2

10.1101/091256 ◽

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.

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A trans-membrane segment inside the ribosome exit tunnel triggers RAMP4 recruitment to the Sec61p translocase

The Journal of Cell Biology ◽

10.1083/jcb.200807066 ◽

2009 ◽

Vol 185 (5) ◽

pp. 889-902 ◽

Cited By ~ 36

Author(s):

Martin R. Pool

Keyword(s):

Endoplasmic Reticulum ◽

Membrane Protein ◽

Ribosomal Protein ◽

Exit Site ◽

Signaling Function ◽

Exit Tunnel ◽

Ribosome Exit Tunnel ◽

A Site

Membrane protein integration occurs predominantly at the endoplasmic reticulum and is mediated by the translocon, which is formed by the Sec61p complex. The translocon binds to the ribosome at the polypeptide exit site such that integration occurs in a cotranslational manner. Ribosomal protein Rpl17 is positioned such that it contacts both the ribosome exit tunnel and the surface of the ribosome near the exit site, where it is intimately associated with the translocon. The presence of a trans-membrane (TM) segment inside the ribosomal exit tunnel leads to the recruitment of RAMP4 to the translocon at a site adjacent to Rpl17. This suggests a signaling function for Rpl17 such that it can recognize a TM segment inside the ribosome and triggers rearrangements of the translocon, priming it for subsequent TM segment integration.

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The ribosome can discriminate the chirality of amino acids within its peptidyl-transferase center

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1424712112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6038-6043 ◽

Cited By ~ 45

Author(s):

Michael T. Englander ◽

Joshua L. Avins ◽

Rachel C. Fleisher ◽

Bo Liu ◽

Philip R. Effraim ◽

...

Keyword(s):

Amino Acids ◽

Physiological Role ◽

Peptidyl Transferase ◽

Translational Machinery ◽

Peptidyl Transferase Center ◽

Peptidyl Transfer ◽

Site Use ◽

Dynamics Simulations ◽

A Site ◽

Trna Binding

The cellular translational machinery (TM) synthesizes proteins using exclusively L- or achiral aminoacyl-tRNAs (aa-tRNAs), despite the presence of D-amino acids in nature and their ability to be aminoacylated onto tRNAs by aa-tRNA synthetases. The ubiquity of L-amino acids in proteins has led to the hypothesis that D-amino acids are not substrates for the TM. Supporting this view, protein engineering efforts to incorporate D-amino acids into proteins using the TM have thus far been unsuccessful. Nonetheless, a mechanistic understanding of why D-aa-tRNAs are poor substrates for the TM is lacking. To address this deficiency, we have systematically tested the translation activity of D-aa-tRNAs using a series of biochemical assays. We find that the TM can effectively, albeit slowly, accept D-aa-tRNAs into the ribosomal aa-tRNA binding (A) site, use the A-site D-aa-tRNA as a peptidyl-transfer acceptor, and translocate the resulting peptidyl-D-aa-tRNA into the ribosomal peptidyl-tRNA binding (P) site. During the next round of continuous translation, however, we find that ribosomes carrying a P-site peptidyl-D-aa-tRNA partition into subpopulations that are either translationally arrested or that can continue translating. Consistent with its ability to arrest translation, chemical protection experiments and molecular dynamics simulations show that P site-bound peptidyl-D-aa-tRNA can trap the ribosomal peptidyl-transferase center in a conformation in which peptidyl transfer is impaired. Our results reveal a novel mechanism through which D-aa-tRNAs interfere with translation, provide insight into how the TM might be engineered to use D-aa-tRNAs, and increase our understanding of the physiological role of a widely distributed enzyme that clears D-aa-tRNAs from cells.

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