Influence of duplexes 3' to the mRNA initiation codon on the efficiency of monosome formation

The structural features of mRNA molecules that determine their relative translational rates are at present poorly defined. An early and potentially rate-limiting step in this process is the assembly of an intact 80S ribosome at the translational initiation codon. It is generally assumed that the efficiency of this reaction is controlled by structures in the 5' nontranslated region and in the immediate proximity of the AUG initiation codon. In this paper, we present an assay of initial monosome formation and measure the effects of hybridizing mRNA to complementary DNA fragments on the efficiency of this reaction. This hybridization serves to block specific regions of the mRNA from sequence-specific and intramolecular (secondary structure) interactions. We find that cDNAs that block the 5' nontranslated region, the initiation codon, or regions immediately 3' to the initiation codon markedly inhibit 80S ribosome attachment. These results are consistent with previous studies by ourselves and others which suggest that the introduction of secondary structures into this region can result in decreased translational efficiency. In addition, however, we note that cDNAs that hybridize to segments of the coding region significant distances (as many as several hundred bases) 3' to the initiation codon can also inhibit initial ribosome binding. This effect appears to be limited to duplexes within the mRNA coding region since a cDNA hybridizing exclusively within the 3' nontranslated region does not inhibit, and may actually stimulate, monosome formation. The results of this monosome formation assay therefore suggest that mRNA structures remote from the 5' terminus and initiation codon may also be important in determining the efficiency of translational initiation.