Destabilization of codon-anticodon interaction in the ribosomal exit site.

The affinities of the exit (E) site of poly(U) or poly(A)-programmed Escherichia coli ribosomes for the respective cognate tRNA and a number of non-cognate tRNAs were determined by equilibrium titrations. Among the non-cognate tRNAs, the binding constants vary up to about tenfold (10(6) to 10(7) M-1 at 20 mM-Mg2+) or 50-fold (10 mM-Mg2+), indicating that codon-independent binding is modulated to a considerable extent by structural elements of the tRNA molecules other than the anticodon. Codon-anticodon interaction stabilizes tRNA binding in the E site approximately fourfold (20 mM-Mg2+) or 20-fold (10 mM-Mg2+), corresponding to delta G degree values of -3 and -7 kJ/mol (0.7 and 1.7 kcal/mol), respectively. Thus, the energetic contribution of codon-anticodon interaction to tRNA binding in the E site appears rather small, particularly in comparison to the large effects on the binding in A and P sites and to the binding of complementary oligonucleotides or of tRNAs with complementary anticodons. This result argues against a role of the E site-bound tRNA in the fixation of the mRNA on the ribosome. In contrast, we propose that the role of the E site is to facilitate the release of the discharged tRNA during translocation by providing an intermediate, labile binding site for the tRNA leaving the P site. The lowering of both affinity and stability of tRNA binding accompanying the transfer of the tRNA from the P site to the E site is predominantly due to the labilization of the codon-anticodon interaction.