The allosteric three-site model for the ribosomal elongation cycle. Analysis with a heteropolymeric mRNA

Ribosomal tRNA binding studies and functional tests were performed at 6 mM Mg2+ using the mRNA analogue C17AUGA4C17 which contains three unique codons in its central region. The following results were obtained. 1) The relative binding affinities of 20 different deacylated tRNAs to nonprogrammed 70 S ribosomes were assessed and were found to vary substantially. 2) When added as the first tRNA, fMet-tRNA and deacylated tRNAs (but not N-acetylated aminoacyl-tRNAs) can bind to internal codons of the mRNA and are therefore suitable for setting the reading frame via codon-anticodon interaction in the peptidyl-tRNA site (P site). 3) After prefilling the P site with deacylated tRNA, the exit site for deacylated tRNA (E site) can be quantitatively occupied only if the cognate codon is present at that site. 4) The translocation of peptidyl-tRNA from the aminoacyl-tRNA site (A site) to the P site is not accompanied by a release of deacylated tRNA. The codon sequence excludes a release and rebinding of deacylated tRNA to the newly exposed A site. Rather, the deacylated tRNA is cotranslocated from the P to the E site where it remains stably bound. 5) After one round of elongation, addition of an A site ligand triggers the dissociation of deacylated tRNA from the E site. Conversely, E site occupation reduces the affinity of the A site for N-acetylated aminoacyl-tRNA. Thus, A and E sites are allosterically linked via negative cooperativity. The results support the allosteric three-site model as an appropriate description of the ribosomal elongation cycle.