Binding of transfer ribonucleic acid to ribosomes engaged in protein synthesis: Number and properties of ribosomal binding sites

Abstract

The results reported in this paper suggest a three-site model for the interaction of S-RNA with active ribosomes. (1) The entrance or decoding site, which requires the integrity of the active complex, admits only a charged S-RNA matching the messenger codon. In step with the GTP-dependent advancement of the messenger tape, this newly selected aminoacyl-S-RNA becomes linked to the end of the nascent polypeptide chain as it moves into (2) the middle or condensing site which is located on the 50 s subunit. At the same time, the preceding S-RNA is discharged and displaced into (3) the exit site. Consequently, with each round of peptide bond formation, a discharged S-RNA molecule is ejected from the exit site, and the entrance site cleared for the admission of a new charged S-RNA molecule specified by the messenger codon. This conclusion is based on the following observations. Using ergosomes (polyribosomes) from rat liver and purified [32P]S-RNA charged with labeled leucine, it was found that each active ribosome binds in the average at least two and at most three S-RNA molecules present in three distinct states: (α) charged with amino acid, (β) attached to the end of the nascent polypeptide chain, and (γ) without amino acid. Aminoacyl-charged and peptide-linked S-RNA molecules are tightly bound and not removable by washing or exchange; their uptake is irreversible and requires GTP and transfer enzymes. By contrast, 1^he uncharged S-RNA is removable by washing and, in the absence of the components necessary for peptide bond formation (GTP, transfer enzymes), equilibrates with uncharged but not with charged S-RNA in the medium. Under optimal conditions of amino acid incorporation, each ribosome contains on the average close to one peptide-linked and one uncharged S-RNA molecule, but less than 0·5 in the aminoacyl form. During controlled dissociation of ergosomes into ribosomal subunits, aminoacyl-charged S-RNA is released from the active complex, whereas the polypeptide-linked S-RNA appears in association with an unstable 56 s particle that subsequently changes into the less compact 50 s subunit with concomitant loss of S-RNA-linked nascent protein. Release of nascent protein with puromycin does not affect S-RNA binding.