The yeast eIF3 subunits TIF32/a, NIP1/c, and eIF5 make critical connections with the 40S ribosome in vivo.
Initiation factor 3 (eIF3) forms a multifactor complex (MFC) with eIF1, eIF2, and eIF5 that stimulates Met-tRNA(i)(Met) binding to 40S ribosomes and promotes scanning or AUG recognition. We have previously characterized MFC subcomplexes produced in vivo from affinity-tagged eIF3 subunits lacking discrete binding domains for other MFC components. Here we asked whether these subcomplexes can bind to 40S ribosomes in vivo. We found that the N- and C-terminal domains of NIP1/eIF3c, the N- and C-terminal domains of TIF32/eIF3a, and eIF5 have critical functions in 40S binding, with eIF5 and the TIF32-CTD performing redundant functions. The TIF32-CTD interacted in vitro with helices 16-18 of domain I in 18S rRNA, and the TIF32-NTD and NIP1 interacted with 40S protein RPS0A. These results suggest that eIF3 binds to the solvent side of the 40S subunit in a way that provides access to the interface side for the two eIF3 segments (NIP1-NTD and TIF32-CTD) that interact with eIF1, eIF5, and the eIF2/GTP/Met-tRNA(i)(Met) ternary complex.
Valásek
Leos
L
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development/NIH, Bethesda, MD 20892, USA.
Mathew
Amy A
AA
Shin
Byung-Sik
BS
Nielsen
Klaus H
KH
Szamecz
Béla
B
Hinnebusch
Alan G
AG
D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5 / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D014176:Protein Biosynthesis; D017434:Protein Structure, Tertiary; D012337:RNA, Ribosomal, 18S / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism
D018797:Cell Cycle Proteins; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins; C077915:NIP1 protein, S cerevisiae; D009687:Nuclear Proteins; D012337:RNA, Ribosomal, 18S; C111889:RPG1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins