title: Eukaryotic translation initiation factor 3 plays distinct roles at the mRNA entry and exit channels of the ribosomal preinitiation complex.
abstract: Eukaryotic translation initiation factor 3 (eIF3) is a central player in recruitment of the pre-initiation complex (PIC) to mRNA. We probed the effects on mRNA recruitment of a library ofS. cerevisiaeeIF3 functional variants spanning its 5 essential subunits using an in vitro-reconstituted system. Mutations throughout eIF3 disrupt its interaction with the PIC and diminish its ability to accelerate recruitment to a native yeast mRNA. Alterations to the eIF3a CTD and eIF3b/i/g significantly slow mRNA recruitment, and mutations within eIF3b/i/g destabilize eIF2•GTP•Met-tRNAibinding to the PIC. Using model mRNAs lacking contacts with the 40S entry or exit channels, we uncovered a critical role for eIF3 requiring the eIF3a NTD, in stabilizing mRNA interactions at the exit channel, and an ancillary role at the entry channel requiring residues of the eIF3a CTD. These functions are redundant: defects at each channel can be rescued by filling the other channel with mRNA.
authors: Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors: Colin Echeverría
authors: CE
authors: Aitken
authors: Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Prague, Czech Republic.
authors: Petra
authors: P
authors: Beznosková
authors: Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Prague, Czech Republic.
authors: Vladislava
authors: V
authors: Vlčkova
authors: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors: Wen-Ling
authors: WL
authors: Chiu
authors: Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors: Fujun
authors: F
authors: Zhou
authors: Laboratory of Regulation of Gene Expression, Institute of Microbiology ASCR, Prague, Czech Republic.
authors: Leoš Shivaya
authors: LS
authors: Valášek
authors: Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors: Alan G
authors: 0000-0002-1627-8395
authors: AG
authors: Hinnebusch
authors: Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, United States.
authors: Jon R
authors: 0000-0002-4521-4999
authors: JR
authors: Lorsch
mesh_terms: D004252:DNA Mutational Analysis; D039621:Eukaryotic Initiation Factor-3 / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D014176:Protein Biosynthesis; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
chemical_list: D039621:Eukaryotic Initiation Factor-3; D050505:Mutant Proteins; D021122:Protein Subunits; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate
keywords: S. cerevisiae; biochemistry; biophysics; eIF3; initiation; mRNA recruitment; ribosome; structural biology; translation; yeast