Abstract
Chloramphenicol (CHL) and linezolid (LZD) are antibiotics that inhibit translation. Both were thought to block peptide bond formation between all combinations of amino acids. Yet recently, a strong nascent peptide context-dependency of CHL- and LZD-induced translation arrest was discovered. Here, we probed the mechanism of action of CHL and LZD by using single-molecule Förster resonance energy transfer spectroscopy (smFRET) to monitor translation arrest induced by antibiotics. The presence of CHL or LZD does not significantly alter dynamics of protein synthesis until the arrest-motif of the nascent peptide is generated. Inhibition of peptide-bond formation compels the fully accommodated A-site tRNA to undergo repeated rounds of dissociation and non-productive rebinding. The glycyl amino-acid moiety on the A-site Gly-tRNA manages to overcome the arrest by CHL. Our results illuminate the mechanism of CHL and LZD action through their interactions with the ribosome, the nascent peptide and the incoming amino acid, perturbing elongation dynamics.
Highlights
Many protein synthesis inhibitors act by sterically disrupting the association and/or positioning of the substrates in the PTC and blocking peptide bond formation[12,13,14,15]
We show that the ability of Gly-tRNAGly to overcome translation stall is determined by the nature of its aminoacyl moiety rather than the tRNAGly body, highlighting the exceptional properties of glycine as a peptidyl acceptor in the CHL-arrested ribosome
Based on the results of our previous Ribo-seq analysis[23] of the effects of CHL and LZD, we designed three model mRNAs encoding peptides with the sequences MFKAFKNIIRTRTL, MFKYFKNIIRTRTL and MFKAFGNIIRTRTL for use in smFRET experiments
Summary
Many protein synthesis inhibitors act by sterically disrupting the association and/or positioning of the substrates in the PTC and blocking peptide bond formation[12,13,14,15]. CHL is a long-known antibiotic initially isolated from Streptomyces venezuelae[16], whereas LZD is a newer synthetic drug and the first representative of the oxazolidinone class of ribosome inhibitors approved for clinical use[17] Despite their structural differences, both CHL and LZD bind to the same site within the PTC5,18–21, where they are expected to clash with the A-site aminoacyl-moiety on the aa-tRNA. The mechanism of translation rescue by the A-site Gly-tRNAGly specific for CHL is unclear, as potentially both the glycine amino acid and the glycine-specific tRNA may contribute to overcoming the antibiotic action It is unknown whether CHL and LZD stabilize specific conformational and compositional states of an actively translating ribosome conducive to translation arrest. We show that the ability of Gly-tRNAGly to overcome translation stall is determined by the nature of its aminoacyl moiety rather than the tRNAGly body, highlighting the exceptional properties of glycine as a peptidyl acceptor in the CHL-arrested ribosome
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