Biochemical Analysis of the eIF2βγ Complex Reveals a Structural Function for eIF2α in Catalyzed Nucleotide Exchange

125

Eukaryotic and archaeal initiation factors 2 (e/aIF2) are heterotrimeric proteins (␣␤␥) carrying methionylated initiator tRNA to the small subunit of the ribosome. The three-dimensional structure of aIF2␥ from the Archaea Pyrococcus abyssi was previously solved. This subunit forms the core of the heterotrimer. The ␣ and ␤ subunits bind the ␥, but do not interact together. aIF2␥ shows a high resemblance with elongation factor EF1-A. In this study, we characterize the role of each subunit in the binding of the methionylated initiator tRNA. Studying various aminoacyl-tRNA ligands shows that the methionyl group is a major determinant for recognition by aIF2. aIF2␥ alone is able to specifically bind Met-tRNA i Met , although with a reduced affinity as compared with the intact trimer. Site-directed mutagenesis confirms a binding mode of the tRNA molecule similar to that observed with the elongation factor. Under our assay conditions, aIF2␤ is not involved in the docking of the tRNA molecule. In contrast, aIF2␣ provides the heterotrimer its full tRNA binding affinity. Furthermore, the isolated C-domain of aIF2␣ is responsible for binding of the ␣ subunit to ␥. This binding involves an idiosyncratic loop of domain 2 of aIF2␥. Association of the C-domain of aIF2␣ to aIF2␥ is enough to retrieve the binding affinity of tRNA for aIF2. The N-terminal and central domains of aIF2␣ do not interfere with tRNA binding. However, the N-domain of aIF2␣ interacts with RNA unspecifically. Based on this property, a possible contribution of aIF2␣ to formation of a productive complex between aIF2 and the small ribosomal subunit is envisaged.In the initiation of translation, the recognition of a start codon on a mRNA involves a specialized initiator tRNA. This tRNA is always esterified with methionine. Several proteins, called initiation factors, contribute along with the ribosome to the success of the initiator tRNA-mRNA interaction. Such initiation factors differ in the three domains of life.

Section: Discussionmentioning

confidence: 62%

“…This serine is conserved in all eucaryal eIF2␣ sequences, not in the archaeal aIF2␣ ones. In eucarya, the eIF2␣ subunit is thought to play an important role in the control of the action of the nucleotide exchange factor eIF2B (31). In Archaea, eIF2B has no equivalent, and nucleotide exchange on aIF2 is thought to occur spontaneously.…”

mentioning

confidence: 99%

Functional Molecular Mapping of Archaeal Translation Initiation Factor 2

Yatime¹,

Schmitt

Blanquet

et al. 2004

125

“…A recent paper by Nika and co-authors (18) has shown that unphosphorylated eIF2␣ also meditates nucleotide exchange. The rate of eIF2B-catalyzed nucleotide exchange increases in the absence of the ␣-subunit compared with wild type, which suggests that nucleotide exchange requires direct interaction between eIF2␣ and eIF2B and that phosphorylation strengthens this interaction.…”

Section: Eukaryotic Translation Initiation Factor 2 (Eif2)mentioning

confidence: 99%

Crystal Structure of the N-terminal Segment of Human Eukaryotic Translation Initiation Factor 2α

Nonato¹,

Widom

Clardy

2002

Eukaryotic translation initiation factor 2␣ (eIF2␣) is a member of the eIF2 heterotrimeric complex that binds and delivers Met-tRNA iMet to the 40 S ribosomal subunit in a GTP-dependent manner. Phosphorylation/dephosphorylation of eIF2␣ at Ser-51 is the major regulator of protein synthesis in eukaryotic cells. Here, we report the first structural analysis on eIF2, the three-dimensional structure of a 22-kDa N-terminal portion of human eIF2␣ by x-ray diffraction at 1.9 Å resolution. This structure contains two major domains. The N terminus is a ␤-barrel with five antiparallel ␤-strands in an oligonucleotide binding domain (OB domain) fold. The phosphorylation site (Ser-51) is on the loop connecting ␤3 and ␤4 in the OB domain. A helical domain follows the OB domain, and the first helix has extensive interactions, including a disulfide bridge, to fix its orientation with respect to the OB domain. The two domains meet along a negatively charged groove with highly conserved residues, indicating a likely site for protein-protein interaction. Eukaryotic translation initiation factor 2 (eIF2)1 is a GTPbinding protein that plays a central role in initiating translation. It binds charged initiator tRNA (Met-tRNA i Met ) in a GTPdependent manner to form the ternary complex, eIF2⅐GTP⅐Met-tRNA iMet . This ternary complex binds to the 40 S ribosomal subunit, and additional initiation factors (including eIF3 and eIF1A) join to form the 43 S preinitiation complex and assist in recognizing the start codon (reviewed in Refs. 1-5).Recognition of the translational site is accompanied by eIF5-mediated hydrolysis of eIF2-bound GTP, which releases an eIF2⅐GDP binary complex along with several other initiation factors. For another round of initiation, the GDP in the eIF2 binary complex must be exchanged for GTP in a reaction catalyzed by the multimeric protein factor eIF2B (previously called guanine nucleotide exchange factor) (6). The 40 S⅐mRNA⅐Met-tRNA i Met complex joins the 60 S ribosomal subunit to form an 80 S initiation complex that can enter the elongation phase of protein synthesis.In eukaryotes, eIF2 is a heterotrimer composed of ␣ (36 kDa)-, ␤ (38 kDa)-, and ␥ (52 kDa)-subunits, which appear to remain associated throughout the initiation cycle. Cross-linking and genetic studies have suggested that both ␤-and ␥-subunits are implicated in guanine nucleotide and Met-tRNA i Met binding (7-9). In addition, the ␤-subunit was shown to interact specifically with eIF5 during GTP hydrolysis and also to bind mRNA (10 -12). The ␥-subunit participates in the recognition of the start site for protein synthesis (13).The phosphorylation/dephosphorylation of a conserved serine (Ser-51) in the ␣-subunit is the major regulator of protein synthesis in eukaryotes (reviewed in Ref.2). Phosphorylation of Ser-51 shuts off protein synthesis (14). Three protein kinases, HRI (heme-regulated inhibitor), PKR (RNA-dependent protein kinase), and GCN2, have been identified that specifically phosphorylate Ser-51 in response to a variety of cellular stresses...

“…Met binding (33), and the lethal phenotype associated with loss of eIF2␣ in yeast is partially suppressed by overproduction of Met-tRNA i Met , eIF2␤, and eIF2␥ (34). Further supporting the idea that eIF2␣ contributes to Met-tRNA i Met binding by eIF2␥, we found that overexpression of either eIF2␣ or Met-tRNA i Met partially suppressed the slow growth phenotype of a yeast strain expressing the eIF2␥-V402A/D403A/L406A triple mutant (Fig.…”

Section: D Middle Panel)mentioning

confidence: 99%

X-ray Structure of Translation Initiation Factor eIF2γ

Roll-Mecak

Alone

Cao

et al. 2004

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...