Interactions of eukaryotic translation initiation factor 3 (eIF3) subunit NIP1/c with eIF1 and eIF5 promote preinitiation complex assembly and regulate start codon selection.
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 of the NIP1-NTD 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 preinitiation complexes and also regulates the selection of AUG start codons in vivo.
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA. valasekl@biomed.cas.cz
Leos
L
Valásek
Klaus H
KH
Nielsen
Fan
F
Zhang
Christie A
CA
Fekete
Alan G
AG
Hinnebusch
D000409:Alanine / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D018387:Codon, Initiator / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D015977:Eukaryotic Initiation Factor-1 / Q000378:metabolism*; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5 / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006160:Guanosine Triphosphate / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D039665:Prokaryotic Initiation Factor-3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D014176:Protein Biosynthesis / Q000235:genetics; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000378:metabolism; D012358:RNA, Transfer, Met / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D018387:Codon, Initiator; D004268:DNA-Binding Proteins; D015977:Eukaryotic Initiation Factor-1; D039621:Eukaryotic Initiation Factor-3; D040061:Eukaryotic Initiation Factor-5; C077915:NIP1 protein, S cerevisiae; D009687:Nuclear Proteins; D039665:Prokaryotic Initiation Factor-3; D021122:Protein Subunits; D012358:RNA, Transfer, Met; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate; D011494:Protein Kinases; D000409:Alanine