Efficient incorporation of eukaryotic initiation factor 1 into the multifactor complex is critical for formation of functional ribosomal preinitiation complexes in vivo.
Eukaryotic initiation factor 1 (eIF1) is a low molecular weight factor critical for stringent AUG selection in eukaryotic translation. It is recruited to the 43 S complex in the multifactor complex (MFC) with eIF2, eIF3, and eIF5 via multiple interactions with the MFC constituents. Here we show that FLAG epitope tagging of eIF1 at either terminus abolishes its in vitro interactions with eIF5 and eIF2beta but not that with eIF3c. Nevertheless, both forms of FLAG-eIF1 fail to bind eIF3 and are incorporated into the 43 S complex inefficiently in vivo. C-terminal FLAG tagging of eIF1 is lethal; overexpression of C-terminal FLAG-eIF1 severely impedes 43 S complex formation and derepresses GCN4 translation due to limiting of eIF2.GTP.Met-tRNA(i)(Met) ternary complex binding to the ribosome. Furthermore, N-terminal FLAG-eIF1 overexpression reduces eIF2 binding to the ribosome and moderately derepresses GCN4 translation. Our results provide the first in vivo evidence that eIF1 plays an important role in promoting 43 S complex formation as a core of factor interactions. We propose that the coordinated recruitment of eIF1 to the 40 S ribosome in the MFC is critical for the production of functional 40 S preinitiation complex.
Singh
Chingakham Ranjit
CR
Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, Kansas 66506, USA.
He
Hui
H
Ii
Miki
M
Yamamoto
Yasufumi
Y
Asano
Katsura
K
D001483:Base Sequence; D004271:DNA, Fungal / Q000235:genetics; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040061:Eukaryotic Initiation Factor-5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D046911:Macromolecular Substances; D010442:Peptide Chain Initiation, Translational; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D015977:Eukaryotic Initiation Factor-1; D015852:Eukaryotic Initiation Factor-2; D040061:Eukaryotic Initiation Factor-5; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins