Release of initiation factors from 48S complexes during ribosomal subunit joining and the link between establishment of codon-anticodon base-pairing and hydrolysis of eIF2-bound GTP

Abstract

The 40S subunit in 48S complexes formed at the initiation codon of mRNA is bound to eukaryotic initiation factor (eIF) 3, eIF1, eIF1A, and an eIF2/GTP/Met-tRNA Met i ternary complex and can therefore not join a 60S subunit directly to form an 80S ribosome. We report that eIF5-induced hydrolysis of eIF2-bound GTP in 48S complexes led to release of eIF2-GDP but not eIF3 or eIF1. eIF5B did not influence factor release in the absence of 60S subunits. Therefore eIF3 and eIF1 dissociate from 40S subunits during, rather than before, the eIF5B-mediated subunit joining event. In the absence of eIF1, eIF5-stimulated hydrolysis of eIF2-bound GTP occurred at the same rate in 43S pre-initiation and 48S initiation complexes. GTP hydrolysis in 43S complexes assembled with eIF1 was much slower than in 43S or 48S complexes assembled without eIF1. Establishment of codon-anticodon base-pairing in 48S complexes relieved eIF1's inhibition. Thus, in addition to its role in initiation codon selection during 48S complex formation, eIF1 also participates in maintaining the fidelity of the initiation process at a later stage, hydrolysis of eIF2-bound GTP, by inhibiting premature GTP hydrolysis and by linking establishment of codon-anticodon base-pairing with GTP hydrolysis.[Keywords: Eukaryotic initiation factor 2; eukaryotic initiation factor 3; eukaryotic initiation factor 5; eukaryotic initiation factor 5B; GTP hydrolysis; translation] Supplemental material is available at http://www.genesdev.org.

Structure of eIF3b RNA Recognition Motif and Its Interaction with eIF3j

“…Similarly, human eIF3 complex lacking eIF3j and eIF3 subcomplexes consisting of eIF3abgi or only eIF3bgi also require eIF3j to stably associate with 40 S subunits (8,28). Here we also demonstrate that the isolated eIF3b-RRM requires eIF3j to bind stably to 40 S subunits (Fig.…”

“…In accordance, high concentrations of salt disrupted the eIF3b-RRM⅐eIF3j complex during NMR titration experiments (data not shown). The eIF3j binding to the eIF3 complex displays a similar salt dependence and dissociates from the rest of eIF3 during sucrose density gradient centrifugation in buffer containing 400 mM KCl (28).…”

“…Although eIF3j is a component of a binary eIF3⅐40 S complex, it is no longer associated with eIF3 and the 40 S subunit in the presence of mRNA as well as in 48 S complexes (4,28). In such ribosomal particles lacking eIF3j, interactions of eIF3a, eIF3b, and eIF3d with mRNA (28) might then instead support the stable association of eIF3 with the 40 S subunit and thereby replace the requirement of eIF3j for stable eIF3/40 S binding.…”

“…Additionally, the yeast homologue, which shares 26% identity and 50% similarity with human eIF3b, also fails to bind RNA in vitro (27). Within 48 S complexes (28) or in binary internal ribosome entry site RNA⅐eIF3 complexes (29), RNA binding of mammalian full-length eIF3b has been evidenced by cross-linking to mRNA, indicating that eIF3b can interact with RNA only in the context of eIF3 complexes. Our data suggest that the ␤-sheet surface of the eIF3b-RRM is an unlikely candidate for mediating the aforementioned RNA cross-links.…”

Coupled Release of Eukaryotic Translation Initiation Factors 5B and 1A from 80S Ribosomes following Subunit Joining

“…In addition, the majority of eIF2 either dissociates from or repositions on the 48S complex following AUG codon recognition (32). Intriguingly, factors eIF3, eIF1, and eIF1A appear to be retained on the 48S complex following eIF2 release (38).…”