Transfer RNA selection at the ribosomal A and P sites

Abstract

We have examined ribosomal selectivity among tRNAs using an equilibrium competition binding assay. Specifically, we evaluated the dissociation constants of tRNA Phe, tRNA Ile, tRNA fMet, tRNA Val, tRNA Arg, and tRNA Glu, for the A site and, separately, for the P site by measuring competition with [ 3H]phenyl-lactyl-tRNA Phe. We were able to distinguish A and P sites using the site-specific poisons, edeine and tetracycline. In the presence of poly(U), the affinity of tRNA Phe for the P site was significantly greater than that of any of the other five tRNAs. Moreover, the affinities of the tRNAs for the P site varied over three orders of magnitude and paralleled the predicted order of affinities based on potential codon-antidocon interactions. This strongly indicates coding capacity at the P site. In marked contrast, tRNA binding at the A site of poly(U)-programmed ribosomes indicated that tRNA Phe bound only ten- to 20-fold stronger than several non-complementary tRNAs, suggesting that the equilibrium properties of the bare ribosomal A site are very different from the P site. We propose that, in vivo, ribosomal contributions to the accuracy of protein synthesis include both specificity in tRNA selection at the A site and the coding capacity of the P site, as these sites are defined by in vitro criteria. Even at best, however, the precision of the ribosomes of the three strains we have examined is not better than the precision of unaided codon-anticodon interactions. This sets new constraints on the sources of accuracy in protein synthesis. We have also measured the relative affinities of tRNAs for the P site in the absence of message. By comparison of these with the codon-instructed P site, we conclude that message confers an additional affinity for a tRNA possessing a matching anticodon, rather than strongly rejecting tRNAs whose codons do not match. All the data we have are consistent with a large, almost non-specific affinity for tRNAs in both P and A sites, to which a smaller specific contribution is made by codon-anticodon base-pairing.