The Binding of Non-cognate Tyr-tRNATyr to Poly(uridylic acid)-Programmed Escherichia coli Ribosomes

Publisher Website
Google Scholar
Abstract
The poly(U)-dependent binding of Tyr-tRNATyr to E. coli ribosomes was studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNATyr/ribosome), and requires the presence of elongation factor (EF) T (a mixture of EF-Ts and EF-Tu), GTP, NH4+ ions and an aminoglycoside antibiotic (streptomycin, neomycin B, kanamycin B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, 1 molecule of GTP is hydrolyzed/molecule of Tyr-tRNATyr bound, suggesting that proof-reading mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNATyr apparently takes place at the ribosomal acceptor site, since peptide bonds are readily formed with N-acetyl-Phe-tRNA prebound to the ribosomal donor site. In contrast to Phe-tRNAPhe binding is impaired by the omission of the 50-S subunit, the replacement of GTP by its non-hydrolyzable analogs guanyl-5''-yl methylene diphosphonate and guanyl-5''-yl iminodiphosphonate, and also by the presence of the antibiotic streptogramin A. This suggests that the correct interaction of Tyr-tRNAtyr with the peptidyl transferase center is essential for the stability of this ligand on the ribosome. The aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNATyr on the ribosome.