Abstract
The first step in translation initiation consists in the recruitment of the small ribosome onto the mRNA. This preinitiation complex (PIC) loads via interactions with eIF4F that has assembled on the 5' cap. It then scans the 5' TL (transcript leader) to locate a start site. The molecular architecture of the PIC-mRNA complex over the cap is beginning to be resolved. As part of this, we have been examining the role of the 5' TL length. We observed in vivo initiation events on AUG codons positioned within 3 nts of the 5' cap and robust initiation in vitro at start sites immediately downstream of the 5' end. Ribosomal toe-printing confirmed the positioning of these codons within the P site, indicating that the ribosome reads from the +1 position. To explore differences in the eIF4E-5' cap interaction in the context of long versus short TL, we followed the fate of the eIF4E-cap interaction using a novel solid phase in vitro expression assay. We observed that ribosome recruitment onto a short TL disrupts the eIF4E-cap contact releasing all the mRNA from the solid phase, whereas with a long the mRNA distributes between both phases. These results are discussed in the context of current recruitment models.