Crystal Structure of the Archaeal Translation Initiation Factor 2 in Complex with a GTP Analogue and Met-tRNA_f^Met

Abstract

Heterotrimeric aIF2αβγ (archaeal homologue of the eukaryotic translation initiation factor 2) in its GTP-bound form delivers Met-tRNA_i^Met to the small ribosomal subunit. It is known that the heterodimer containing the GTP-bound γ subunit and domain 3 of the α subunit of aIF2 is required for the formation of a stable complex with Met-tRNA_i. Here, the crystal structure of an incomplete ternary complex including aIF2αD3γ ⋅ GDPNP ⋅ Met-tRNA_f^Met has been solved at 3.2 Å resolution. This structure is in good agreement with biochemical and hydroxyl radical probing data. The analysis of the complex shows that despite the structural similarity of aIF2γ and the bacterial translation elongation factor EF-Tu, their modes of tRNA binding are very different. Remarkably, the recently published 5.0-Å-resolution structure of almost the same ternary initiation complex differs dramatically from the structure presented. Reasons for this discrepancy are discussed.

Introduction

In eukaryotic and archaeal cells, translation initiation factor 2 (e/aIF2) in its GTP-bound form specifically binds Met-tRNA_i^Met (tRNA_i) and delivers it to the small ribosomal subunit. After recognition of the start codon, this factor in its GDP-bound form loses affinity for tRNA_i and subsequently dissociates from the initiation complex. e/aIF2 is a heterotrimeric protein, consisting of α, β, and γ subunits. The e/aIF2α subunit domain structure is conserved in Eukarya and Archaea,[1], [2], [3], [4] while the β subunit is only half of its eukaryotic counterpart by mass and is homologous to its C-terminal part. The γ subunit, which is the largest of the subunits, forms the core of the heterotrimer and interacts with both the α and β subunits, whereas the α and β subunits do not interact with each other.[4], [5], [6], [7] The e/aIF2γ sequences are conserved in Archaea and mammals,⁸ suggesting that they share a common three-dimensional structure. Moreover, archaeal aIF2 has been shown to correctly place initiator Met-tRNA_i into the P site of the mammalian 40S ribosomal subunit and accompany the entire set of eukaryotic translation initiation factors in the process of cap-dependent scanning and AUG codon selection.⁹

However, despite strong structural similarity between e/aIF2 from the two domains of life, the archaeal and eukaryotic IF2 factors show several differences in the tRNA_i and guanine nucleotide binding sites.⁶ Thus, in eIF2, all subunits contribute to the binding of tRNA_i,¹⁰ whereas in Archaea, only the α and γ subunits are required for the formation of a stable complex with tRNA_i.[6], [11] Moreover, it has been found that the α subunit can be replaced by its C-terminal domain (αD3) in a functional αγ heterodimer.¹¹

The γ subunit of aIF2 is structurally related to the translation elongation factor EF-Tu⁵ and consists of three domains. Domain I (the G domain) contains the nucleotide-binding pocket and two functionally essential flexible regions, switch 1 and switch 2, while domains II and III are β-barrels. The α subunit also consists of three domains, which will be referred to as domains 1, 2 and 3, to distinguish them from the γ subunit domains.

Currently, two structures of the bacterial translation elongation factor EF-Tu in complex with the GTP analogue GDPNP and tRNA are available.[12], [13] In both complexes, the protein interacts with tRNA through the acceptor stem and T stem–loop. These structures were subsequently used as templates to build aIF2 ⋅ GTP ⋅ Met-tRNA_i^Met models,[3], [14], [15] which, however, could only provide a partial explanation of available biochemical data related to aIF2–tRNA_i interactions. Recently, a 5.0-Å-resolution structure of the aIF2αβγ ⋅ GDPNP ⋅ Met-tRNA_f^Met complex from Sulfolobus solfataricus was published.¹⁶ In this structure, tRNA_f^Met was found to contact the α and γ subunits through the elbow and minor groove of the acceptor stem. Remarkably, the RNA–protein contacts and the relative arrangement of the protein and tRNA_f^Met in this structure did not agree with earlier biochemical[6], [11] and hydroxyl radical probing experiments.¹⁵

Here, we present the crystal structure of the aIF2 from S. solfataricus, containing the γ subunit and domain 3 of the α subunit (SsoaIF2αD3γ) in complex with Escherichia coli Met-tRNA_f^Met at 3.2 Å resolution. The overall shape and domain arrangement of the γ subunit in this complex have not been described previously. In contrast to the structure of the ternary complex EF-Tu ⋅ GDPNP ⋅ Phe-tRNA^Phe, in the structure presented, Met-tRNA_f^Met contacts the protein through the acceptor stem, D stem–loop, and the CCA-Met end, which is positioned between the G domain and domain III of the γ subunit. In contrast to the earlier structure of the complex,¹⁶ the structure presented here is in good agreement with biochemical and hydroxyl radical probing data. We discuss possible reasons for the discrepancy between the two structures.