Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA

Abstract

Heterotrimeric eukaryotic/archaeal translation initiation factor 2 (e/aIF2) binds initiator methionyl-tRNA and plays a key role in the selection of the start codon on messenger RNA. tRNA binding was extensively studied in the archaeal system. The γ subunit is able to bind tRNA, but the α subunit is required to reach high affinity whereas the β subunit has only a minor role. In Saccharomyces cerevisiae however, the available data suggest an opposite scenario with β having the most important contribution to tRNA-binding affinity. In order to overcome difficulties with purification of the yeast eIF2γ subunit, we designed chimeric eIF2 by assembling yeast α and β subunits to archaeal γ subunit. We show that the β subunit of yeast has indeed an important role, with the eukaryote-specific N- and C-terminal domains being necessary to obtain full tRNA-binding affinity. The α subunit apparently has a modest contribution. However, the positive effect of α on tRNA binding can be progressively increased upon shortening the acidic C-terminal extension. These results, together with small angle X-ray scattering experiments, support the idea that in yeast eIF2, the tRNA molecule is bound by the α subunit in a manner similar to that observed in the archaeal aIF2-GDPNP-tRNA complex.

Figure 1.

Eukaryotic and archaeal e/aIF2. (A) Cartoon representation of Ss-aIF2 in complex with GDPNP and Met-tRNA_f^Met. The cartoon was drawn from PDB ID 3V11 (15). The color code is as follows: G-domain of γ (γDI, 1–210) in green, domain II (γDII, 211–327) in yellow, domain III (γDIII, 328–415) in orange, domain 1 of α in dark blue (αD1, 1–85), domain 2 of α in blue (αD2, 86–174), domain 3 of α in cyan (αD3, 175–266). The N-terminal α helix of the β subunit (3–19) anchored to γDI is colored in pink. Note that the position of the rest of the β subunit (residues 33–139, encircled with a dotted line) is only a tentative model, derived from SAXS data (15). The figure was drawn with PyMOL (http://www.pymol.org). (B) Schematic structural organizations of e/aIF2 α and β subunits. The colored boxes indicate the structural domains. Specific eukaryotic domains and extensions are colored in orange. Gray bars symbolize the K-boxes in the N-terminal domain of yeast eIF2β.

Figure 2.

SDS–PAGE analysis of purified Ch-eIF2 variants. The 12.5% SDS–PAGE were stained with Coomassie Blue. Lane 1, Ch-eIF2 heterotrimer. Lane 2, Ch-eIF2αγ heterodimer. Lane 3, Ch-eIF2βγ heterodimer. Lane 4, Ch-eIF2(αΔC274)βγ. Positions of tagged yeast eIF2α (Y-α, 36·9 kDa), tagged yeast eIF2αΔC274 (Y-αΔC, 33·4 kDa), yeast eIF2β (Y-β, 31·4 kDa) and Ss-aIF2γ (Ss-γ, 45·6 kDa) are indicated. Lane 5, Ch-eIF2βΔNΔCγ heterodimer. Lane 6, Ch-eIF2βΔNγ heterodimer. Lane 7, Ch-eIF2αΔC274γ heterodimer. Positions of yeast eIF2βΔNΔC (Y-βΔNΔC), eIF2βΔN (Y-βΔN) and eIF2αΔC274 (Y-αΔC) are indicated. Lane 8, ChEncc-eIF2(αΔC274)γ heterodimer, Lane 9, ChEncc-eIF2αγ heterodimer. Positions of tagged yeast eIF2αΔC274 (Y-αΔC, 33·4 kDa), tagged yeast eIF2α (Y-α, 36·9 kDa) and E. cuniculi eIF2γ (Encc−γ, 48·9 kDa) are indicated.

Figure 3.

Cloverleaf representations of initiator Met-tRNAs from S. solfataricus, E. coli and S. cerevisiae cytoplasm. Nucleotides denoted with gray boxes are universally conserved in initiator tRNAs. Post-transcriptional modifications are indicated in the cases of E. coli and S. cerevisiae.

Figure 4.

Alignment of e/aIF2α domain 3 sequences. Representative eukaryotic (upper block) and archaeal (lower block) sequences are shown. Aspartate and glutamate residues are boxed in gray. Secondary structures are drawn below the Ss-aIF2α sequence. The positions of the last residue in C-terminal truncated versions of eIF2α are indicated.