scholarly journals Structural basis for context-specific inhibition of translation by oxazolidinone antibiotics

2021 ◽  
Author(s):  
Kaitlyn Tsai ◽  
Vanja Stojković ◽  
D. John Lee ◽  
Iris D. Young ◽  
Teresa Szal ◽  
...  
Keyword(s):  
Peptide Bond ◽  
Structural Basis ◽  
Side Chain ◽  
Specific Inhibition ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Nascent Chain ◽  
Context Specific ◽  
Small Hydrophobic ◽  
Penultimate Position

The antibiotic linezolid, the first clinically approved member of the oxazolidinone class, inhibits translation of bacterial ribosomes by binding to the peptidyl transferase center. Recent work has demonstrated that linezolid does not inhibit peptide bond formation at all sequences but rather acts in a context-specific manner, namely when alanine occupies the penultimate position of the nascent chain. In this study, we determined that the second-generation oxazolidinone radezolid also induces stalling with alanine at the penultimate position. However, the molecular basis for context-specificity of these inhibitors has not been elucidated. In this study, we determined high-resolution cryo-EM structures of both linezolid and radezolid-stalled ribosome complexes. These structures reveal that the alanine side chain fits within a small hydrophobic crevice created by oxazolidinone, resulting in improved ribosome binding. Modification of the ribosome by the antibiotic resistance enzyme Cfr disrupts stalling by forcing the antibiotic to adopt a conformation that narrows the hydrophobic alanine pocket. Together, the structural and biochemical findings presented in this work provide molecular understanding of context-specific inhibition of translation by clinically important oxazolidinone antibiotics.

scholarly journals Structural basis for the context-specific action of classic peptidyl transferase inhibitors.

2021 ◽  
Author(s):  
Egor A. Syroegin ◽  
Laurin Flemmich ◽  
Dorota S Klepacki ◽  
Nora S Vazquez-Laslop ◽  
Ronald Micura ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Molecular Mechanisms ◽  
Peptide Bond ◽  
Amino Acid Sequences ◽  
Structural Basis ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Inhibit Protein Synthesis ◽  
Clinically Significant ◽  
Context Specific

Ribosome-targeting antibiotics serve both as powerful antimicrobials and as tools for studying the ribosome. The ribosomal catalytic site, the peptidyl transferase center (PTC), is targeted by a large number of various drugs. The classical and best-studied PTC-acting antibiotic chloramphenicol, as well as the newest clinically significant linezolid, were considered indiscriminate inhibitors of every round of peptide bond formation, presumably inhibiting protein synthesis by stalling ribosomes at every codon of every gene being translated. However, it was recently discovered that chloramphenicol or linezolid, and many other PTC-targeting drugs, preferentially arrest translation when the ribosome needs to polymerize particular amino acid sequences. The molecular mechanisms and structural bases that underlie this phenomenon of context-specific action of even the most basic ribosomal antibiotics, such as chloramphenicol, are unknown. Here we present high-resolution structures of ribosomal complexes, with or without chloramphenicol, carrying specific nascent peptides that support or negate the drug action. Our data suggest that specific amino acids in the nascent chains directly modulate the antibiotic affinity to the ribosome by either establishing specific interactions with the drug molecule or obstructing its placement in the binding site. The model that emerged from our studies rationalizes the critical importance of the penultimate residue of a growing peptide for the ability of the drug to stall translation and provides the first atomic-level understanding of context specificity of antibiotics that inhibit protein synthesis by acting upon the PTC.

scholarly journals Ribosome crystallography: catalysis and evolution of peptide-bond formation, nascent chain elongation and its co-translational folding

2005 ◽  
Vol 33 (3) ◽  
pp. 488-492 ◽  
Author(s):  
A. Bashan ◽  
A. Yonath
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Trigger Factor ◽  
Chain Elongation ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Chemical Catalysis ◽  
Nascent Chain ◽  
Peptidyl Transferase Centre ◽  
Folding Space

A ribosome is a ribozyme polymerizing amino acids, exploiting positional- and substrate-mediated chemical catalysis. We showed that peptide-bond formation is facilitated by the ribosomal architectural frame, provided by a sizable symmetry-related region in and around the peptidyl transferase centre, suggesting that the ribosomal active site was evolved by gene fusion. Mobility in tunnel components is exploited for elongation arrest as well as for trafficking nascent proteins into the folding space bordered by the bacterial chaperone, namely the trigger factor.

scholarly journals Context-specific inhibition of translation by ribosomal antibiotics targeting the peptidyl transferase center

2016 ◽  
Vol 113 (43) ◽  
pp. 12150-12155 ◽  
Author(s):  
James Marks ◽  
Krishna Kannan ◽  
Emily J. Roncase ◽  
Dorota Klepacki ◽  
Amira Kefi ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Peptide Bond ◽  
Bacterial Cells ◽  
Peptidyl Transferase ◽  
Drug Induced ◽  
Translation Arrest ◽  
Peptidyl Transferase Center ◽  
Nascent Chain ◽  
Context Specific

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.

scholarly journals Structural Basis of the Ribosomal Machinery for Peptide Bond Formation, Translocation, and Nascent Chain Progression

2003 ◽  
Vol 11 (1) ◽  
pp. 91-102 ◽  
Author(s):  
Anat Bashan ◽  
Ilana Agmon ◽  
Raz Zarivach ◽  
Frank Schluenzen ◽  
Joerg Harms ◽  
...  
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Structural Basis ◽  
Peptide Bond Formation ◽  
Nascent Chain

A Possible Mechanism of Peptide Bond Formation on Ribosome without Mediation of Peptidyl Transferase

1999 ◽  
Vol 200 (2) ◽  
pp. 193-205 ◽  
Author(s):  
GOURAB KANTI DAS ◽  
DHANANJAY BHATTACHARYYA ◽  
DEBI PROSAD BURMA
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase

On Ribosome Conservation and Evolution

2006 ◽  
Vol 52 (3-4) ◽  
pp. 359-374 ◽  
Author(s):  
Ilana Agmon ◽  
Anat Bashan ◽  
Ada Yonath
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Binding Modes ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Peptide Bonds ◽  
Peptidyl Transferase Center ◽  
Enzymatic Function ◽  
Rotatory Motion ◽  
Rna Fold

The ribosome is a ribozyme whose active site, the peptidyl transferase center (PTC), is situated within a highly conserved universal symmetrical region that connects all ribosomal functional centers involved in amino acid polymerization. The linkage between this elaborate architecture and A-site tRNA position revealed that the A-> P-site passage of the tRNA terminus in the peptidyl transferase center is performed by a rotatory motion, synchronized with the overall tRNA/mRNA sideways movement. Guided by the PTC, the rotatory motion leads to stereochemistry suitable for peptide bond formation, as well as for substrate-mediated catalysis, consistent with quantum mechanical calculations elucidating the transition state mechanism for peptide bond formation and indicating that the peptide bond is being formed during the rotatory motion. Analysis of substrate binding modes to inactive and active ribosomes illuminated the significant PTC mobility and supported the hypothesis that the ancient ribosome produced single peptide bonds and non-coded chains, utilizing free amino acids. Genetic control of the reaction evolved after poly-peptides capable of enzymatic function were created, and an ancient stable RNA fold was converted into tRNA molecules. As the symmetry relates only the backbone fold and nucleotide orientations, but not nucleotide sequence, it emphasizes the superiority of functional requirement over sequence conservation, and indicates that the PTC has evolved by gene fusion, presumably by taking advantage of similar RNA fold structures.

A Competitive Model Reaction for the Ribosomal Peptide Bond Formation Catalyzed by Peptidyl Transferase

2014 ◽  
Vol 496-500 ◽  
pp. 17-20
Author(s):  
Lin Cheng ◽  
Nian Hong ◽  
Xiang Qun Xu ◽  
Jie Yang ◽  
You Quan Zhong
Keyword(s):  
Reaction Pathway ◽  
Bond Formation ◽  
Peptide Bond ◽  
Model Reaction ◽  
Reaction Pathways ◽  
Peptide Bond Formation ◽  
Peptidyl Transferase ◽  
Reaction Field ◽  
Self Consistent ◽  
Competitive Model

In this work, a series of theoretical methods were employed to investigate the reaction mechanisms of ribosomal peptide bond formation catalyzed by peptidyl transferase. For the studies described in this paper, reaction pathways and free energy barriers for the model reaction of the peptide bond synthesis were studied by performing Ab initio calculation. Two self-consistent reaction field (SCRF) methods were used to calculate of the whole reaction pathway. These results show that the present theoretical reaction mechanism is a potential and competitive one for the reaction modeling of the ribosomal peptide synthesis.

scholarly journals Prevention, by ribosome-bound nascent polyphenylalanine chains, of the functional interaction of t-2 toxin with its receptor site

1976 ◽  
Vol 156 (2) ◽  
pp. 289-294 ◽  
Author(s):  
M Cannon ◽  
K E Smith ◽  
C J Carter
Keyword(s):  
Critical Time ◽  
Receptor Site ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Inhibitory Effects ◽  
Peptidyl Transferase ◽  
Amino Acid Incorporation ◽  
Functional Interaction ◽  
Time Period ◽  
Critical Chain

1. The inhibitory effects of T-2 toxin and trichodermin on poly(U)-directed polyphenylalanine synthesis were studied by using cell-free systems from reticulocytes. Conditions for amino acid incorporation were carefully chosen in an attempt to ensure that the large majority of poly(U) chains bound only one ribosome engaged in protein synthesis and that all such ribosomes carried nascent polyphenylalanine chains containing approximately the same number of residues. 2. Cell-free systems were allowd to synthesize polyphenylalanine, and T-2 toxin and trichodermin were added to the incorporation mixtures at various times. Irrespective of the time of addition, trichodermin (50 μg/ml) inhibited polyphenylalanine synthesis by approx. 70%. In contrast, although T-2 toxin (40 μg/ml), when added at early incubation times, could inhibit polyphenylalanine synthesis with a maximum of 50%, the drug had no effect on the system when added after a critical time-period. 3. It is concluded that although both T-2 toxin and trichodermin can inhibit peptide-bond formation on ribosomes at the level of the peptidyl transferase catalytic centre the presence, on ribosomes, of nascent polyphenylalanine chains above a certain critical chain length excludes T-2 toxin from functional interaction with its receptor site.

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