Sequential eukaryotic translation initiation factor 5 (eIF5) binding to the charged disordered segments of eIF4G and eIF2beta stabilizes the 48S preinitiation complex and promotes its shift to the initiation mode

During translation initiation in Saccharomyces cerevisiae, an Arg- and Ser-rich segment (RS1 domain) of eukaryotic translation initiation factor 4G (eIF4G) and the Lys-rich segment (K-boxes) of eIF2 bind three common partners, eIF5, eIF1, andmRNA. Here, we report that both of these segments are involved in mRNA recruitment and AUG recognition by distinct mechanisms. First, the eIF4G-RS1 interaction with the eIF5 C-terminal domain (eIF5-CTD) directly links eIF4G to the preinitiation complex (PIC) and enhances mRNA binding. Second, eIF2-K-boxes increase mRNA binding to the 40S subunit in vitro in a manner re-versed by the eIF5-CTD. Third, mutations altering eIF4G-RS1, eIF2-K-boxes, and eIF5-CTD restore the accuracy of start codon selection impaired by an eIF2mutation in vivo, suggesting that the mutual interactions of the eIF segments within the PIC prime the ribosome for initiation in response to start codon selection. We propose that the rearrangement of interactions involv-ing the eIF5-CTD promotes mRNA recruitment throughmRNA binding by eIF4G and eIF2 and assists the start codon-induced release of eIF1, the major antagonist of establishing tRNAi Met:mRNA binding to the P site. In eukaryotic translation, initiation factors (eIFs) promote dis-sociation of vacant 80S (canonical initiation) or posttermina-tion (recycling) ribosomes and assist binding ofMet-tRNAi Met and 5=-capped mRNA to the 40S subunit to form 43S and 48S preini