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

Abstract

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-tRNA^Phe for the A and P sites of 80S ribosomes and the cooperativity constant were measured at different temperature and Mg²⁺ concentration. The AcPhe-tRNA^Phe 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-tRNA^Phe 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.

Abbreviations

tRNA^Phe

deacylated phenylalanine tRNA

Phe-tRNA^Phe

phenylalanyl-tRNA^Phe

AcPhe-tRNA^Phe

N-acetyl-phenylalanyl-tRNA^Phe

tRNA^-Phe

total tRNA preparation free of phenylalanine tRNA

v̄^Σ

the average number of tRNA molecules bound per ribosome or ribosomal subunit