title: Human eukaryotic initiation factor 2 (eIF2)-GTP-Met-tRNAi ternary complex and eIF3 stabilize the 43 S preinitiation complex.
abstract: The formation of a stable 43 S preinitiation complex (PIC) must occur to enable successful mRNA recruitment. However, the contributions of eIF1, eIF1A, eIF3, and the eIF2-GTP-Met-tRNAi ternary complex (TC) in stabilizing the 43 S PIC are poorly defined. We have reconstituted the human 43 S PIC and used fluorescence anisotropy to systematically measure the affinity of eIF1, eIF1A, and eIF3j in the presence of different combinations of 43 S PIC components. Our data reveal a complicated network of interactions that result in high affinity binding of all 43 S PIC components with the 40 S subunit. Human eIF1 and eIF1A bind cooperatively to the 40 S subunit, revealing an evolutionarily conserved interaction. Negative cooperativity is observed between the binding of eIF3j and the binding of eIF1, eIF1A, and TC with the 40 S subunit. To overcome this, eIF3 dramatically increases the affinity of eIF1 and eIF3j for the 40 S subunit. Recruitment of TC also increases the affinity of eIF1 for the 40 S subunit, but this interaction has an important indirect role in increasing the affinity of eIF1A for the 40 S subunit. Together, our data provide a more complete thermodynamic framework of the human 43 S PIC and reveal important interactions between its components to maintain its stability.
authors.affiliation: Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California 95616.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Sokabe
authors.affiliation: Department of Molecular and Cellular Biology, College of Biological Sciences, University of California, Davis, California 95616. Electronic address: csfraser@ucdavis.edu.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Fraser
mesh_terms: D016880:Anisotropy; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry*; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry*; D006160:Guanosine Triphosphate / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012358:RNA, Transfer, Met / Q000737:chemistry*; D054682:Ribosome Subunits, Small, Eukaryotic / Q000737:chemistry; D012270:Ribosomes / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
chemical_list: D015852:Eukaryotic Initiation Factor-2; D039621:Eukaryotic Initiation Factor-3; D012358:RNA, Transfer, Met; D006160:Guanosine Triphosphate
keywords: Cooperativity; Eukaryotic Translation Initiation; Protein-Protein Interaction; RNA-binding Protein; Ribosome; Ribosome Function; eIF1; eIF1A; eIF2; eIF3