Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes

TLDR

Flanking sequences around the initiator codon in eukaryotic mRNAs are non‑random. The study tests whether purines at positions –3 or +4 aid AUG recognition by performing in vitro binding assays and proposes a modified scanning mechanism. In vitro binding assays using 32P‑labeled oligonucleotides were performed to evaluate ribosome interaction, and a modified scanning mechanism was suggested. Among 153 examined mRNAs, 151 displayed a purine at –3 or a G at +4, establishing the consensus A/GXXAUGG for functional sites, and in vitro assays showed that purines at these positions markedly enhance ribosome binding, supporting a modified scanning model.

Abstract

Sequences flanking the initiator codon in eukaryotic mRNAs are not random. Out of 153 messages examined, 151 have either a purine in position -3, or a G in position +4, or both. Thus, [A/G]XXAUGG emerges as the favored sequence for eukaryotic initiation sites. Nucleotides flanking nonfunctional AUG triplets, which occur in the 5'-noncoding region of a few eukaryotic messages, are different from those found at most functional sites. Whereas most authentic initiator codons are preceded by a purine (usually A) in position -3, most nonfunctional AUGs have a pyrimidine in that position. The observed asymmetry suggests that purines in positions -3 and +4 might facilitate recognition of the AUG condon during formation of initiation complexes. To test this idea, in vitro binding studies were carried out with 32P-labeled oligonucleotides. Binding of AUG-containing oligonucleotides to wheat germ ribosomes was significantly enhanced by placing a purine in position -3 or +4. The scanning model, which postulates that 40S ribosomal subunits attach at the 5'-end of a message and migrate down to the AUG codon, is discussed in light of these new observations. A modified version of the scanning mechanism is proposed.