INITIAL BINDING OF THE ELONGATION-FACTOR U-CENTER-DOT-GTP-CENTER-DOT-AMINOACYL-TRANSFER-RNA COMPLEX PRECEDING CODON RECOGNITION ON THE RIBOSOME

The first step in the sequence of interactions between the ribosome an d the complex of elongation factor Tu (EF-Tu), GTP, and aminoacyl-tRNA , which eventually leads to A site-bound aminoacyl-tRNA, is the codon- independent formation of an initial complex. We have characterized the initial binding and the resulting complex by time resolved (stopped-f low) and steady-state fluorescence measurements using several fluoresc ent tRNA derivatives. The complex is labile, with rate constants of 6 x 10(7) M(-1) s(-1) and 24 s(-1) (20 degrees C, 10 mM Mg2+) for bindin g and dissociation, respectively. Both thermodynamic and activation pa rameters of initial binding were determined, and five Mg2+ ions were e stimated to participate in the interaction. While a cognate ternary co mplex proceeds from initial binding through codon recognition to rapid GTP hydrolysis, the rate constant of GTP hydrolysis in the non-cognat e complex is 4 orders of magnitude lower, despite the rapid formation of the initial complex in both cases, Hence, the ribosome-induced GTP hydrolysis by EF-Tu is strongly affected by the presence of the tRNA. This suggests that codon-anticodon recognition, which takes place afte r the formation of the initial binding complex, provides a specific si gnal that triggers fast GTP hydrolysis by EF-Tu on the ribosome.