Fidelity of the eukaryotic codon-anticodon interaction: interference by aminoglycoside antibiotics.
A homologous in vitro method was developed from Tetrahymena for ribosomal A-site binding of aminoacyl-tRNA to poly(uridylic acid)-programmed ribosomes with very low error frequency. The reaction mixture pH was the crucial factor in the stable A-site association of aminoacyl-tRNA with high fidelity. At a pH greater than 7.1, endogenous activity translocated A-site-bound aminoacyl-tRNA to the P site. If translocation was allowed to occur, a near-cognate amino-acyl-tRNA, Leu-tRNA, could stably bind to the ribosome by translocation to the ribosomal P site. Near-cognate aminoacyl-tRNA did not stably bind to either site when translocation was blocked. Misreading antibiotics stimulated the stable association of near-cognate aminoacyl-tRNA to the ribosomal A site, thereby increasing the error frequency by several orders of magnitude. Ribosome binding of total aminoacyl-tRNA near equilibrium was not inhibited by misreading antibiotics; however, initial rate kinetics of the binding reaction were dramatically altered such that a 6-fold rate increase was observed with paromomycin or hygromycin B. The rate increase was evident with both cognate and near-cognate aminoacyl-tRNAs. Several antibiotics were tested for misreading potency by the ribosome binding method. We found gentamicin G418 greater than paromomycin greater than neomycin greater than hygromycin B greater than streptomycin in the potentiation of misreading. Tetracycline group antibiotics effectively inhibited A-site aminoacyl-tRNA binding without promoting misreading.
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000926:Anticodon; D001665:Binding Sites; D003062:Codon; D006863:Hydrogen-Ion Concentration; D010445:Peptide Elongation Factors / Q000378:metabolism; D014176:Protein Biosynthesis / Q000187:drug effects*; D012270:Ribosomes / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013752:Tetracycline / Q000494:pharmacology; D013768:Tetrahymena; D046249:Transfer RNA Aminoacylation
D000900:Anti-Bacterial Agents; D000926:Anticodon; D003062:Codon; D010445:Peptide Elongation Factors; D013752:Tetracycline