Interface Between 40s Exit Channel Protein Us7/Rps5 And Eif2 Alpha Modulates Start Codon Recognition In Vivo

The eukaryotic pre-initiation complex (PIC) bearing the eIF2.GTP.Met-tRNA(i)(Met) ternary complex (TC) scans the mRNA for an AUG codon in favorable context. AUG recognition evokes rearrangement of the PIC from an open, scanning to a closed, arrested conformation. Cryo-EM reconstructions of yeast PICs suggest remodeling of the interface between 40S protein Rps5/uS7 and eIF2 alpha between open and closed states; however, its importance was unknown. uS7 substitutions disrupting eIF2 alpha contacts favored in the open complex increase initiation at suboptimal sites, and uS7-S223D stabilizes TC binding to PICs reconstituted with a UUG start codon, indicating inappropriate rearrangement to the closed state. Conversely, uS7-D215 substitutions, perturbing uS7-eIF2 alpha interaction in the closed state, confer the opposite phenotypes of hyperaccuracy and (for D215L) accelerated TC dissociation from reconstituted PICs. Thus, remodeling of the uS7/eIF2 alpha interface appears to stabilize first the open, and then the closed state of the PIC to promote accurate AUG selection in vivo.