Binding of wheat germ ribosomes to bisulfite-modified reovirus messenger RNA: Evidence for a scanning mechanism

A scanning mechanism has been proposed (Kozak, 1978) to explain how eukaryotic ribosomes select the correct AUG codon for initiation of protein synthesis. The hypothesis is that a 40 S ribosomal subunit binds initially at or near the 5′-terminus of a message and subsequently migrates toward the interior of the messenger RNA, stopping when it encounters the first AUG codon, at which point a 60 S subunit joins and peptide bond formation begins. The scanning mechanism predicts that if a message were modified by introduction of a new AUG triplet upstream of the existing initiator codon, the adventitious AUG should be the preferred site for formation of an 80 S initiation complex. This prediction has been confirmed in the present studies with two reovirus messenger RNAs, in which sodium bisulfite was used to convert an ACG sequence (located in the 5′ untranslated region of each message) to AUG. Analysis of the ribosome-protected mRNA fragments recovered from sparsomycin-blocked 80 S initiation complexes revealed that a high percentage of wheat germ ribosomes were centered around the “unnatural” 5′-proximal AUG created by the bisulfite treatment, although some ribosomes were also positioned at the second (normal) initiator codon. The bisulfite modification was carried out in 7 m-urea at 37 °C. resulting in quantitative conversion of cytosine to uracil. Thus, both the primary and secondary structure of the message were drastically altered. These perturbations did not impair the efficiency of ribosome binding, nor did the highly unfolded state of the mRNA permit ribosomes to attach to spurious sites in the interior of the message. The data support a mechanism in which the initiator codon is selected by virtue of its position in a message (i.e. closest to the 5′-terminus), without regard to either the primary or secondary structure of the flanking regions.