title: Functional reconstitution of human eukaryotic translation initiation factor 3 (eIF3).
abstract: Protein fate in higher eukaryotes is controlled by three complexes that share conserved architectural elements: the proteasome, COP9 signalosome, and eukaryotic translation initiation factor 3 (eIF3). Here we reconstitute the 13-subunit human eIF3 in Escherichia coli, revealing its structural core to be the eight subunits with conserved orthologues in the proteasome lid complex and COP9 signalosome. This structural core in eIF3 binds to the small (40S) ribosomal subunit, to translation initiation factors involved in mRNA cap-dependent initiation, and to the hepatitis C viral (HCV) internal ribosome entry site (IRES) RNA. Addition of the remaining eIF3 subunits enables reconstituted eIF3 to assemble intact initiation complexes with the HCV IRES. Negative-stain EM reconstructions of reconstituted eIF3 further reveal how the approximately 400 kDa molecular mass structural core organizes the highly flexible 800 kDa molecular mass eIF3 complex, and mediates translation initiation.
authors.affiliation: California Institute for Quantitative Biosciences, University of California, Berkeley, CA 94720, USA.
authors.forename: Chaomin
authors.initials: C
authors.lastname: Sun
authors.forename: Aleksandar
authors.initials: A
authors.lastname: Todorovic
authors.forename: Jordi
authors.initials: J
authors.lastname: Querol-Audí
authors.forename: Yun
authors.initials: Y
authors.lastname: Bai
authors.forename: Nancy
authors.initials: N
authors.lastname: Villa
authors.forename: Monica
authors.initials: M
authors.lastname: Snyder
authors.forename: John
authors.initials: J
authors.lastname: Ashchyan
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Lewis
authors.forename: Abbey
authors.initials: A
authors.lastname: Hartland
authors.forename: Scott
authors.initials: S
authors.lastname: Gradia
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Fraser
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Doudna
authors.forename: Eva
authors.initials: E
authors.lastname: Nogales
authors.forename: Jamie H D
authors.initials: JH
authors.lastname: Cate
mesh_terms: D000075686:COP9 Signalosome Complex; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3 / Q000737:chemistry*; D006367:HeLa Cells; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008854:Microscopy, Electron / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D046912:Multiprotein Complexes / Q000737:chemistry; D010447:Peptide Hydrolases / Q000737:chemistry; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry
chemical_list: D018076:DNA, Complementary; D039621:Eukaryotic Initiation Factor-3; D046912:Multiprotein Complexes; D012333:RNA, Messenger; D010447:Peptide Hydrolases; D000075686:COP9 Signalosome Complex