The Interaction between Eukaryotic Initiation Factor 1A and eIF5 Retains eIF1 within Scanning Preinitiation Complexes

Abstract

Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors including eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using NMR, we unveil eIF1A’s binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A-interaction site. We also found that the interaction between eIF1A:eIF5-CTD is conserved between human and yeast. Using GST pull down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A:eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5

“…8). During mRNA scanning, eIF1A-NTT interaction with the eIF5-CTD helps to retain eIF1 in the PIC (Luna et al, 2013) (Fig. 8a).…”

“…eIF1 maintains the PIC in the open conformation and prevents Met-tRNA i to accommodate in the P-site (P OUT ). eIF1A-NTT also binds eIF5 (Luna et al, 2013), preventing its binding by eIF2β-NTT. The binding partner of eIF2β-NTT at this stage may be eIF1 (Nanda et al, 2013) or alternatively, rRNA or mRNA, as it binds RNA (Singh et al, 2012).…”

“…eIF5 is the GTPase activating protein for eIF2 (Asano et al, 2001b; Huang et al, 1997). Independently of the catalytic NTD, the eIF5 C-terminal domain (CTD) interacts with eIF1A, eIF2β, eIF3c, and eIF4G at various stages of initiation (Luna et al, 2013; Luna et al, 2012; Reibarkh et al, 2008; Singh et al, 2012; Yamamoto et al, 2005). While an initial cryoEM study revealed density potentially corresponding to eIF5-CTD facing eIF1 and eIF2 in the PIC (Hussain et al, 2014), this was not observed in more recent PIC structures (Llacer et al, 2015).…”

Structural insights into the mammalian late-stage initiation complexes

“…Noteworthy, eIF1A NTT was shown to interact with eIF5 36,37 , but because of its clear involvement in the start codon/anticodon duplex, we suggest that this eIF5 interaction occurs during the pre-initiation phase and very shortly after the recruitment of the mRNA.…”

“…2a,b). AUG codons of both mRNAs face the (34) CAU (36) of anticodon stem-loop (ASL) tRNA i Met , within hydrogenbonding distances (~2.7Å). In the case of β-globin mRNA, the codon:anticodon interaction is stabilized further by the N-terminal tail (NTT) of eIF1A (Lys7 interacts with the ribose of G(+3) from mRNA, Fig.…”