Abstract
The carboxyl-terminal domain (CTD) of eukaryotic initiation factor (eIF) 5 interacts with eIF1, eIF2beta, and eIF3c, thereby mediating formation of the multifactor complex (MFC), an important intermediate for the 43 S preinitiation complex assembly. Here we demonstrate in vitro formation of a nearly stoichiometric quaternary complex containing eIF1 and the minimal segments of eIF2beta, eIF3c, and eIF5. In vivo, overexpression of eIF2 and tRNA(Met)(i) suppresses the temperature-sensitive phenotype of tif5-7A altering eIF5-CTD by increasing interaction of the mutant eIF5 with eIF2 by mass action and restoring its defective interaction with eIF3. By contrast, overexpression of eIF1 exacerbated the tif5-7A phenotype because eIF1 forms unusual inhibitory complexes with a hyperstoichiometric amount of eIF1. Formation of such complexes leads to increased GCN4 translation, independent of eIF2 phosphorylation (general control derepressed or Gcd(-) phenotype). We also provide biochemical evidence indicating that the association of eIF5-CTD with eIF2beta strongly enhances its binding to eIF3c. Our results suggest strongly that MFC formation is an ordered event involving specific enhancement of eIF5-CTD binding to eIF3 on its binding to eIF2beta. We propose that the primary function of eIF5-CTD is to serve as an assembly guide by rapidly promoting stoichiometric MFC assembly with the aid of eIF2 while excluding formation of nonfunctional complexes.
Highlights
input amount (In) eukaryotic translation initiation, the 40 S ribosomal subunit binds the eukaryotic initiation factor1 21⁄7GTP1⁄7MettRNAiMet ternary complex (TC) to form the 43 S preinitiation complex
Substantial evidence indicates that the carboxyl-terminal domain (CTD) of eIF5 serves as a core of ribosomal preinitiation complex formation (2, 3), independent of its GTPase-activating function ascribed to the amino-terminal domain (NTD) (4, 5)
We attempted to form a quaternary complex with GST-eIF5-B6, His-eIF3c-N, His-eIF2-N, and eIF1-FL that were expressed from bacteria
Summary
AA-boxes 1 and 2 located at the sixth and seventh ␣-helices, and the eighth ␣-helix, respectively (17). Rather the eIF2/eIF5 interaction is the most critical for the functional MFC assembly These results strongly suggest that the entire assembly process is an ordered and highly coordinated event. In support of this idea, we show that the eIF5CTD interaction with eIF2 K-box greatly enhances its binding to eIF3c. Based on these and other findings, we discuss how the ribosomal recruitment of the MFC constituents and their release on AUG recognition are coordinately controlled during the course of translation initiation
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