Interactions of eIF3 Subunit NIP1/c with eIF1 and eIF5 Promote Pre-initiation Complex Assembly and Regulate Start Codon Selection.
Leos Valasek (1), Klaus Nielsen (2), Fan Zhang (2), Christie Fekete (2), Alan Hinnebusch (2)
(1) Division of Cellular and Molecular Microbiology , Institute of Microbiology AS CR, Prague, 142 20, the Czech Republic; (2) Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA
The N-terminal domain (NTD) of NIP1/eIF3c interacts directly with eIF1 and eIF5, and indirectly through eIF5 with the eIF2.GTP.Met-tRNA i Met ternary complex (TC), to form the multifactor complex (MFC). We investigated the physiological importance of these interactions by mutating 16 segments spanning the NIP1-NTD. Mutations in multiple segments reduced the binding of eIF1 or eIF5 to the NIP1-NTD. Mutating a C-terminal segment of the NIP1-NTD increased utilization of UUG start codons (Sui - phenotype) and was lethal in cells expressing eIF5-G31R that is hyperactive in stimulating GTP hydrolysis by the TC at AUG codons. Both effects of this NIP1 mutation were suppressed by eIF1 overexpression, as was the Sui - phenotype conferred by eIF5-G31R. Mutations in two N-terminal segments suppressed the Sui - phenotypes produced by the eIF1-D83G and eIF5-G31R mutations. From these and other findings, we propose that the NIP1-NTD coordinates an interaction between eIF1 and eIF5 that inhibits GTP hydrolysis at non-AUG codons. Two NIP1-NTD mutations were found to derepress GCN4 translation in a manner suppressed by overexpressing the TC, indicating that MFC formation stimulates TC recruitment to 40S ribosomes. Thus, the NIP1-NTD is required for efficient assembly of pre-initiation complexes and also regulates the selection of AUG start codons in vivo.