Structural analysis of an eIF3 subcomplex reveals conserved interactions required for a stable and proper translation pre-initiation complex assembly

Abstract

Translation initiation factor eIF3 acts as the key orchestrator of the canonical initiation pathway in eukaryotes, yet its structure is greatly unexplored. We report the 2.2 Å resolution crystal structure of the complex between the yeast seven-bladed β-propeller eIF3i/TIF34 and a C-terminal α-helix of eIF3b/PRT1, which reveals universally conserved interactions. Mutating these interactions displays severe growth defects and eliminates association of eIF3i/TIF34 and strikingly also eIF3g/TIF35 with eIF3 and 40S subunits in vivo. Unexpectedly, 40S-association of the remaining eIF3 subcomplex and eIF5 is likewise destabilized resulting in formation of aberrant pre-initiation complexes (PICs) containing eIF2 and eIF1, which critically compromises scanning arrest on mRNA at its AUG start codon suggesting that the contacts between mRNA and ribosomal decoding site are impaired. Remarkably, overexpression of eIF3g/TIF35 suppresses the leaky scanning and growth defects most probably by preventing these aberrant PICs to form. Leaky scanning is also partially suppressed by eIF1, one of the key regulators of AUG recognition, and its mutant sui1G107R but the mechanism differs. We conclude that the C-terminus of eIF3b/PRT1 orchestrates co-operative recruitment of eIF3i/TIF34 and eIF3g/TIF35 to the 40S subunit for a stable and proper assembly of 48S pre-initiation complexes necessary for stringent AUG recognition on mRNAs.

Novel insights into the architecture and protein interaction network of yeast eIF3

“…Subjecting Prt1 181C -Tif34-Tif35 to the limited proteolysis abolished the binding of Prt1 to the other two proteins by removing its C-terminal region. This confirms previous observations on the importance of the C-terminal region of Prt1 for binding to Tif34 and Tif35 Valasek et al 2001b, Herrmannova et al 2011. Further analysis of the binary interactions between these three proteins showed that the interaction between Tif35 and Prt1 is weak and sensitive to the salt concentration.…”

“…5A). This is consistent with recent observations indicating that binding of Tif34 to Prt1 markedly stabilizes Tif35 association with the complex (Herrmannova et al 2011). Thermodynamic analysis of the interaction of Tif34 with Tif35 and Prt1…”

“…The solution structure of the RNA recognition motif (RRM) of human eIF3b in complex with a short peptide of eIF3j suggested a noncanonical RRM structure (Elantak et al 2010); however, its yeast ortholog has all the structural features of a canonical RRM (Khoshnevis et al 2010). The recent crystal structure analysis of yeast eIF3i/Tif34 in complex with a short peptide of eIF3b/Prt1 revealed conserved interactions necessary for proper PIC assembly (Herrmannova et al 2011).…”

Novel RNA-binding Protein P311 Binds Eukaryotic Translation Initiation Factor 3 Subunit b (eIF3b) to Promote Translation of Transforming Growth Factor β1-3 (TGF-β1-3)

“…eIF3, the largest eIF complex, is composed of 13 subunits, named eIF3a to eIF3m (30). Several of them have been identified as core subunits (47). eIF3b is one of them, whereas the others are eIF3j, eIF3a, eIF3g, eIF3i, and eIF3e (25,48).…”

“…As such, the eIF3b WD40 domain interacts with the 40 S ribosomal subunit (57). This domain is followed by the C-terminal domain, necessary for the interaction with subunits eIF3i and eIF3g (47,66). Mutational analysis suggests that P311 binding to eIF3b involves the RRM.…”