Interface between 40S exit channel protein uS7/Rps5 and eIF2α modulates start codon recognition in vivo.
The eukaryotic pre-initiation complex (PIC) bearing the eIF2·GTP·Met-tRNAiMetternary 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 eIF2α between open and closed states; however, its importance was unknown. uS7 substitutions disrupting eIF2α 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-eIF2α 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/eIF2α interface appears to stabilize first the open, and then the closed state of the PIC to promote accurate AUG selection in vivo.
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
Jyothsna
J
Visweswaraiah
Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
Alan G
0000-0002-1627-8395
AG
Hinnebusch
D019943:Amino Acid Substitution; D018387:Codon, Initiator / Q000378:metabolism*; D020285:Cryoelectron Microscopy; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism* / Q000648:ultrastructure*; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
D018387:Codon, Initiator; D015852:Eukaryotic Initiation Factor-2; D050505:Mutant Proteins; D012269:Ribosomal Proteins; C030831:ribosomal protein S5
S. cerevisiae; biochemistry; chromosomes; eIF2α; genes; initiation; ribosome; translation; uS7/Rps5; yeast