RESUMO
We have determined several kinetic parameters for the reaction of poly(U)-programmed ribosomes with the near-cognate ternary complex of leucyl-tRNA2, elongation factor Tu (EF-Tu), and GTP. From single-turnover experiments at 5 degrees C with the ribosomes present in excess we find that the apparent second order rate constant for GTP hydrolysis is 0.4 X 10(6) M-1 s-1, that the cleavage step is faster than 4 s-1 and that the apparent rate constant for peptide formation is 6 +/- 3 s-1. From multiple-turnover experiments at 5 degrees C, with the ternary complex present in excess we find that kcat for GTP hydrolysis is 0.4 s-1 and Km is 1.6 microM. For both kinds of experiment the ratio of peptide formed to GTP hydrolyzed is 0.05 +/- 0.2. Comparison of these results with those obtained with the cognate complex show that the ribosome distinguishes between the cognate and near-cognate complexes, not on the basis of the forward rate constants, but on the basis of the reverse and rejection rate constants, which differ for the two complexes by at least 4000- and 100-fold, respectively. Both in ternary complex selection and in proofreading, the frequency of errors observed is much higher than might be expected from these large differences in rate constant. The reason is that, even for the near-cognate species, the rates of ternary complex rejection and aminoacyl-tRNA rejection are not overwhelmingly greater than the forward rate constants for GTP hydrolysis and peptidyl-tRNA formation, respectively. The outcome of these reactions is, therefore, at least partially under kinetic rather than thermodynamic control, leading to the trapping of errors which would not be made in a slow process.
