Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors

The interaction between Escherichia coli 30S ribosomal subunits and mRNAs, and the effect of the initiation factors on this process, have been studied using MS2 RNA, polyribonucleotides and model mRNAs encoded by synthetic genes. The interactions were analyzed by gel filtration, by sucrose gradient centrifugation and by competition for ribosome binding between the various mRNAs and a Shine-Dalgarno deoxyoctanucleotide. It was found that the initiation factors do not significantly affect the Shine-Dalgarno interaction nor the apparent Ka values of the 30S-subunit-mRNA binary complexes, but influence the positioning of the mRNAs on the 30S subunit with respect to the Shine-Dalgarno octanucleotide. The results suggest that, in the absence of initiation factors, the mRNA occupies a ribosomal "stand-by" site which is close to or includes the region where the Shine-Dalgarno interaction takes place; in the presence of the factors, the mRNA is shifted away from the stand-by site, towards another ribosomal site with similar affinity for the mRNA. This shift does not require the presence of fMet-tRNA and, depending upon the type of mRNA, is mediated by IF-2 and/or IF-3.