X-ray Structure of Translation Initiation Factor eIF2γ

Abstract

The x-ray structure of the ␥-subunit of the heterotrimeric translation initiation factor eIF2 has been determined to 2.4-Å resolution. eIF2 is a GTPase that delivers the initiator Met-tRNA to the P site on the small ribosomal subunit during a rate-limiting initiation step in translation. The structure of eIF2␥ closely resembles that of EF1A⅐GTP, consisting of an N-terminal G domain followed by two ␤-barrels arranged in a closed configuration with domain II packed against the G domain in the vicinity of the Switch regions. The G domain of eIF2␥ has an unusual zinc ribbon motif, not previously found in other GTPases. Structure-based site-directed mutagenesis was used to identify two adjacent features on the surface of eIF2␥ that bind the ␣-subunit and Met-tRNA i Met , respectively. These structural, biochemical, and genetic results provide new insights into eIF2 ternary complex assembly.Translation initiation involves assembly of a large protein-RNA complex at the initiation codon of a mRNA in three main steps: binding of initiator Met-tRNA and mRNA with associated factors to the small ribosomal subunit, pairing of the anticodon of Met-tRNA i Met with the AUG start codon, and joining of the two ribosomal subunits to form the 80 S initiation complex. In eukaryotes, this process requires at least twelve distinct translation initiation factors (eIFs) 1 and hydrolysis of two molecules of GTP (reviewed in Ref. 1). Translation initiation factor eIF2 is a heterotrimeric GTPase that delivers the Met-tRNA i Met to the small ribosomal subunit as part of a ternary complex with GTP. Pairing between the anticodon of the Met-tRNA i Met and the AUG start codon triggers hydrolysis of GTP by eIF2 and eIF2⅐GDP is released, leaving Met-tRNA i Met bound in the P site. eIF2⅐GDP cannot bind Met-tRNA i Met and is converted to eIF2⅐GTP by the heteropentameric exchange fac-eIF2 is a stable complex of three subunits (␣, ␤, and ␥), each essential for viability in yeast. Since their discovery, highly conserved eIF2 subunits have been identified in all eukaryotes (reviewed in Ref.2) and also archaebacteria (3). Biochemical and genetic analyses have demonstrated that the three subunits have distinct activities during translation initiation. The ␥-subunit binds GTP and Met-tRNA i Met (reviewed in Ref. 4). GTP hydrolysis by the eIF2 ternary complex bound to the 40 S ribosomal subunit is stimulated by eIF5, which interacts with eIF2␤ (5). The ␤-subunit also interacts with Met-tRNA i Met and has been reported to bind mRNA (6). Given the pivotal role eIF2 plays in eukaryotic translation initiation, it is not surprising that it represents an important target for regulation. In response to various environmental stressors (viral infection, amino acid starvation, heme deficiency, ER stress etc), the ␣-subunit is phosphorylated on Ser 51 , abolishing translation initiation by preventing eIF2B catalyzed GDP-GTP exchange.The ␥-subunit of eIF2 belongs to the superfamily of GTPbinding proteins and is most closely related to the translation elongation factor...

Structure of archaeal translational initiation factor 2 βγ–GDP reveals significant conformational change of the β-subunit and switch 1 region

“…3A). The amphiphilic b␣ 1 -helix interacts with the hydrophobic surface, composed of g␣ 4 , g␤ 6 , g␤ 7 , and g␣ 5 at the back side of the GBS, which comprises an extensive intersubunit hydrophobic core (Fig. 3B).…”

“…The ␥-subunit is the structural core of a͞eIF2 and binds primarily to GTP and Met-tRNA i . It is related structurally to translational EF1A (elongation factor 1A), although the rationale for GTP dependence of Met-tRNA i binding has not been fully understood (4)(5)(6). The N-terminal half of eIF2␤ is characteristic of eukaryotes and associates with RNA (7) and with the initiation factors eIF5 and eIF2B (8), which are also characteristic of eukaryotes.…”

Functional Molecular Mapping of Archaeal Translation Initiation Factor 2

“…It can therefore be proposed that, among the features in domain 2 that distinguish archaeal and eukaryotic a/eIF2 from EF-Tu, the L1 loop is directly involved in the binding of the ␣ subunit. Notably, during revision of the present manuscript, a study concerning aIF2␥ from M. jannaschii was published on line (42). This study shows that aIF2␥-L256D (corresponding to Leu 229 within loop 1 in P. abyssi aIF2, see Fig.…”

Mechanism and Regulation of Protein Synthesis in Saccharomyces cerevisiae

“…The incorporation of eIF2g in the eIF2 complex is dependent on the apparently eIF2-specific chaperone Cdc123 (Perzlmaier et al 2013). The amino acid sequence and structure of eIF2g and aIF2g show striking similarity to elongation factor EF-Tu from bacteria (Hannig et al 1993;Schmitt et al 2002;Roll-Mecak et al 2004). Whereas EF-Tu binds diverse aminoacyl-tRNAs (aa-tRNAs) to the ribosomal A site, eIF2 specifically binds Met-tRNA i Met to the ribosomal P site.…”