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

Abstract

The translation initiation GTPase eukaryotic translation initiation factor 5B (eIF5B) binds to the factor eIF1A and catalyzes ribosomal subunit joining in vitro. We show that rapid depletion of eIF5B in Saccharomyces cerevisiae results in the accumulation of eIF1A and mRNA on 40S subunits in vivo, consistent with a defect in subunit joining. Substituting Ala for the last five residues in eIF1A (eIF1A-5A) impairs eIF5B binding to eIF1A in cell extracts and to 40S complexes in vivo. Consistently, overexpression of eIF5B suppresses the growth and translation initiation defects in yeast expressing eIF1A-5A, indicating that eIF1A helps recruit eIF5B to the 40S subunit prior to subunit joining. The GTPase-deficient eIF5B-T439A mutant accumulated on 80S complexes in vivo and was retained along with eIF1A on 80S complexes formed in vitro. Likewise, eIF5B and eIF1A remained associated with 80S complexes formed in the presence of nonhydrolyzable GDPNP, whereas these factors were released from the 80S complexes in assays containing GTP. We propose that eIF1A facilitates the binding of eIF5B to the 40S subunit to promote subunit joining. Following 80S complex formation, GTP hydrolysis by eIF5B enables the release of both eIF5B and eIF1A, and the ribosome enters the elongation phase of protein synthesis.The initiation of protein synthesis in eukaryotes is a complex series of events orchestrated by initiation factors (eIFs). In vitro studies using purified translation initiation factors have led to the elucidation of the translation initiation pathway in which the different factors associate with the 40S ribosomal subunit and guide the binding of MettRNA i Met and mRNA. While the essential in vitro functions of the different factors are well established, the precise timing of factor binding to and release from the ribosome is less well understood. Moreover, not all of the in vitrodefined functions of the factors have been confirmed in vivo. The first step in translation initiation involves the binding of factor eIF2 to GTP and Met-tRNA i Met forming a ternary complex. The ternary complex along with factors eIF1, eIF1A, eIF3, and eIF5 binds to the 40S ribosome, forming the 43S complex (reviewed in reference 16). The 43S complex then binds to an mRNA near the 5Ј cap, forming a 48S complex that scans along the mRNA in a 3Ј direction in search of an AUG start codon. In the 48S complex, some of the eIF2-GTP is hydrolyzed to eIF2-GDP ϩ P i (2). Upon AUG codon recognition, GTP hydrolysis is completed, and P i is released, converting the 48S complex from an open to a closed complex with Met-tRNA i Met in the P site of the 40S subunit (29). The release of P i from the 48S complex appears to be coupled with displacement of eIF1 from its binding site near the P site (2,22). 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

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…Blocking eIF5B GTPase activity, either by inclusion of nonhydrolyzable GTP analogs or by mutation of the eIF5B G domain, does not impair subunit joining (Shin et al , 2007Acker et al 2009); however, it does impede eIF1A release from 80S ribosomes both in vivo and in vitro (Fringer et al 2007;Acker et al 2009). Mutations that disrupt the GTPase activity of eIF5B severely impair yeast cell growth (Shin et al , 2007.…”

“…Domain IV of eIF5B binds to eIF1A via interaction with the last five residues at the C terminus of eIF1A (Olsen et al 2003;Acker et al 2006;Fringer et al 2007). Mutation of the eIF1A C terminus impairs subunit joining and full activation of eIF5B GTPase activity in vitro (Acker et al 2006) and impairs yeast cell growth and eIF5B binding to 40S complexes in vivo.…”

“…Both eIF1A and eIF5B are bound to the 80S ribosome following subunit joining, and GTP hydrolysis by eIF5B is required for their release Fringer et al 2007;Acker et al 2009). Blocking eIF5B GTPase activity, either by inclusion of nonhydrolyzable GTP analogs or by mutation of the eIF5B G domain, does not impair subunit joining (Shin et al , 2007Acker et al 2009); however, it does impede eIF1A release from 80S ribosomes both in vivo and in vitro (Fringer et al 2007;Acker et al 2009).…”

Eukaryote-specific extensions in ribosomal proteins of the small subunit: Structure and function

“…28,63,64 It also plays a role, along with eIF5B, in ensuring proper subunit joining and formation of elongation-competent 80S ribosomes. 28,63,64 Hydroxyl radical probing and structural modeling involving eIF1A and Tetrahymena thermophila 40S ribosomal subunits (based on bacterial 30S:IF1 complex information) at »3.9A suggested that eIF1A resides near the A-site and makes contacts with eukaryotespecific proteins eS27 and eS30 as well as conserved protein uS12 which harbors eukaryote-specific extensions. 15,65 However, direct involvement of uS12 eukaryote-specific extensions in eIF1A binding has not been proven yet.…”

Translation initiation factors are not required for Dicistroviridae IRES function in vivo

“…Met base-pairs with the AUG, eIF5 stimulates eIF2-GTP hydrolysis, eIF2-GDP is released along with other factors, eIF5B-GTP is recruited to stimulate 60S ribosomal subunit joining, and eIF1A and eIF5B-GDP are released to generate the translationally active 80S complex (Fringer et al 2007;Hinnebusch et al 2007). After each round of initiation, eIF2-GDP must be recycled by eIF2B (a guanine nucleotide exchange factor) for a new round of initiation.…”