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.
Valásek
Leos
L
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892, USA. valasekl@biomed.cas.cz
Nielsen
Klaus H
KH
Zhang
Fan
F
Fekete
Christie A
CA
Hinnebusch
Alan G
AG
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