Interaction of tRNA with the A and P sites of rabbit‐liver 80S ribosomes and their 40S subunits

The interaction between tRNA and rabbit liver 80S ribosomes and 40S subunits was studied using a nitrocellulose membrane filtration technique. Binding of the different tRNA forms (aminoacyl‐, peptidyl‐ or deacylated) to poly(U)‐programmed 40S subunits and 80S ribosomes was found to be a cooperative process. The association constants of AcPhe‐tRNAPhe for the A and P sites of 80S ribosomes and the cooperativity constant were measured at different temperature and Mg2+ concentration. The AcPhe‐tRNAPhe association constant for the P site was shown to be between 2 × 107 M−1 and 2 × 108 M−1 at 25–37°C and 5–20 mM Mg2+, while the affinity for the A site was 10–100‐fold lower. The cooperativity constant was shown to decrease with the increase of incubation temperature and the decrease of Mg2+ concentration. The affinity of AcPhe‐tRNAPhe for the A site of 80S ribosomes was shown to depend upon the codon specificity of tRNA at the P site. The cooperativity of the tRNA interaction with 80S ribosomes was suggested to be mostly contributed by the association with the 40S subunit and result from the correct codon‐anticodon pairing at the P site. The data presented imply a codon‐anticodon interaction at the P site of eukaryotic 80S ribosomes.