A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met.
Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex.
Phan
L
L
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20910, USA.
Schoenfeld
L W
LW
Valásek
L
L
Nielsen
K H
KH
Hinnebusch
A G
AG
D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D005838:Genotype; D007700:Kinetics; D008958:Models, Molecular; D010448:Peptide Initiation Factors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D039665:Prokaryotic Initiation Factor-3; D014176:Protein Biosynthesis; D021122:Protein Subunits; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012358:RNA, Transfer, Met / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D013816:Thermodynamics
D015977:Eukaryotic Initiation Factor-1; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; D005656:Fungal Proteins; D010448:Peptide Initiation Factors; D039665:Prokaryotic Initiation Factor-3; D021122:Protein Subunits; C050998:Prt1 protein, S cerevisiae; D012333:RNA, Messenger; D012358:RNA, Transfer, Met; D029701:Saccharomyces cerevisiae Proteins