Fidelity of the eukaryotic codon-anticodon interaction: interference by aminoglycoside antibiotics

• Published in: Biochemistry (Easton) Vol. 23; no. 7; pp. 1462 - 1467
• Main Authors: Eustice, David C, Wilhelm, James M
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 27.03.1984
• Subjects: Animals; Anti-Bacterial Agents - pharmacology; Anticodon; Binding Sites; Biological and medical sciences; Codon; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Molecular and cellular biology; Molecular genetics; Peptide Elongation Factors - metabolism; Protein Biosynthesis - drug effects; Replication; Ribosomes - metabolism; Structure-Activity Relationship; Tetracycline - pharmacology; Tetrahymena; Tetrahymena thermophila; Transfer RNA Aminoacylation; Fidelity; Antibiotic; Translation; Codon; Ribosome; Streptomycin; Tetrahymena thermophila; Neomycin; Error; Molecular interaction; Anticodon
• DOI: 10.1021/bi00302a019
• ISSN: 0006-2960

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.