SELECTIVITY OF THE A-SITE OF ESCHERICHIA-COLI RIBOSOMES TOWARD DEACYLATED TRANSFER-RNA

The inhibitory effect of several species of E. coli and yeast tRNA(Phe) On formation of the EFTu.GTP [C-14]Phe-tRNA(Phe) and EFTu.GTP [C-14]Lys-tRNA(Lys) complexes was measured to estimate their equilibrium affinity to the A site of E. coli ribosomes. The association constant for codon-specific tRNA and the template measured at 0 degrees C and 10 mM Mg2+ was approximately (1-7).10(-7) M(-1). This value is in agreement with the association constant for factor-independent binding. Removal of two 3'-terminal nucleotides (A76 and C75) had practically no effect on the association constant, implying that they are not involved in nonenzymatic binding to the A site. Among the codon-nonspecific tRNA-template pairs tested, only tRNA(Tyr) and poly(U) had appreciable affinity to the A site (only nine times lower than that for tRNA(Phe)). For other species of tRNA, the affinity was 120-1800 times lower and practically did not correlate with the number of codon-noncomplementary anticodon nucleotides. Thus, the mRNA-programmed A site displays high selectivity toward nonenzymatically bound tRNA, even if the latter is deacylated. This selectivity greatly exceeds the one observed for ribosome-independent codon-anticodon recognition in solution and binding to the P site.