The Binding of Non-cognate Tyr-tRNA&supTyr; to Poly(uridylic acid)-Programmed <em>Escherichia coli</em> Ribosomes.

The poly(U)-dependent binding of Tyr-tRNA^Tyr to Escherichia coli ribosomes has been studied using a highly purified system. Binding is maximal at 10 mM magnesium acetate (up to 0.7 molecule Tyr-tRNA^Tyr/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, kanamycip B or gentamicin C1a). Under limiting and up to saturating concentrations of EF-T, one molecule of GTP is hydrolyzed per molecule of Tyr-tRNA^Tyr bound, suggesting that ‘proof-reading’ mechanisms involving the hydrolysis of GTP are inoperative in the presence of the antibiotics. Binding of Tyr-tRNA^Tyr 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-tRNA^Phe binding, Tyr-tRNA^Tyr 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-tRNA^Tyr with the peptidyl transferase centre is essential for the stability of this ligand on the ribosome. Moreover, the aminoglycoside antibiotics are also necessary, even after the binding reaction is complete, to maintain Tyr-tRNA^Tyr on the ribosome. [ABSTRACT FROM AUTHOR]