Influence of mRNA secondary structure on binding and migration of 40S ribosomal subunits

Reovirus messenger RNA was modified by reaction with bisulfite (in denaturing conditions) or by incorporation of IMP in place of GMP, thereby irreversibly unfolding the mRNA. Messenger RNA in which the secondary structure was weakened or abolished retained the ability to bind to wheat germ ribosomes, suggesting that conformational features around the AUG codon are not required for ribosome recognition of mRNA. Ribosomes were not able to attach (directly) to spurious internal sites, even in extensively unfolded RNA, indicating that the monocistronic character of eucaryotic messages (in which initiation is limited to a single 5' proximal site) is not simply due to conformational masking of all the internal AUG codons. The secondary structure in eucaryotic messages does contribute to the fidelity of the translation process, however, because when 40S ribosomal subunits were incubated with denatured mRNA they failed to stop at the 5' proximal AUG codon. Extensive migration beyond the 5' region occurred when 40S ribosomes (in the absence of 60S subunits) attached to unfolded mRNA, implying that the secondary structure in native mRNA facilitates correct translation by impeding migration of 40S subunits beyond the 5' proximal initiation region. Secondary structure in mRNA may also modulate the efficiency of translation. Studies with BrUMP-substituted mRNA, in which the secondary structure is enhanced, suggested that the efficiency of mRNA binding to ribosomes decreases as the stability of the secondary structure increases.