Journal of Molecular Biology
Binding of the 5′-Triphosphate End of mRNA to the γ-Subunit of Translation Initiation Factor 2 of the Crenarchaeon Sulfolobus solfataricus
Introduction
The heterotrimeric translation initiation factor 2 (e/aIF2) is pivotal for binding and positioning of methionylated initiator tRNA (Met-tRNAi) on the ribosomes of Eukaryotes and Archaea. Like its eukaryotic counterpart eIF2, the archaeal translation initiation factor aIF2 consists of three subunits: α, β and γ. The largest γ-subunit forms the core of the heterotrimer and interacts with both, the α-subunit and the β-subunit, whereas the latter two subunits do not interact with each other. The e/aIF2 γ-subunit contains the G-domain and is a structural homologue of bacterial elongation factor EF-Tu [1]. Archaeal aIF2 has been subjected to extensive crystallographic studies, and the structures of trimeric aIF2 and its γ-subunit have been determined in free and nucleotide-bound states [1], [2], [3], [4], [5], [6], [7], [8], [9], [10]. Two mobile regions of the G-domain, switch 1 and switch 2, are responsible for nucleotide binding and exchange.
The γ-subunit of the crenarchaeon Sulfolobus solfataricus IF2 (SsoIF2γ) serves, besides its central role in Met-tRNAi binding on the ribosome [11], an additional function: SsoIF2γ binds to the 5′-triphosphate end of mRNA and protects its 5′-part from degradation [12]. Similarly, the eukaryotic IF2 has also been shown to interact with mRNA [13], [14]. However, in contrast to the archaeal factor, the eIF2 β-subunit appears to be responsible for mRNA binding [15].
This work was devoted to localization of the mRNA-binding site on the surface of the SsoIF2 γ-subunit. We show that SsoIF2γ binds mRNAs carrying triphosphorylated guanosine at their 5′-end but does not bind mRNAs with adenosine triphosphate at this position. Only GTP and GDP competed with mRNA for binding to SsoIF2γ, whereas other triphosphorylated nucleotides (ATP, UTP and CTP) were unable to do so. Thus, we conclude that mRNAs carrying GTP at the 5′-end bind specifically to SsoIF2γ. Site-directed mutagenesis, crystallographic analyses of wild-type and mutated SsoIF2γ and biochemical studies revealed that the 5′-terminal triphosphorylated guanosine of mRNAs binds to the canonical nucleotide-binding pocket of the SsoIF2 γ-subunit.
Section snippets
Binding of the 5′-triphosphate end of mRNA fragments to SsoIF2γ
As SsoIF2 binding was independent of the nature of mRNAs [12], different RNA fragments were used in these studies (Supplementary Fig. S1). The reason for using the 36-nt and 49 (48)-nt fragments of mRNA encoding archaeal ribosomal protein L1 was that the crystal structures of these fragments are available [16], [17] and that these structures could be used for model building and biochemical data interpretation.
SsoIF2γ was shown to bind exclusively mRNAs with a triphosphate at the 5′-end, whereas
Conclusions and perspective
The biochemical, mutational and structural analyses and MD simulations present strong evidence that the canonical nucleotide-binding pocket of SsoIF2γ is required for binding mRNAs, starting with the 5′-terminal guanosine, which are highly abundant in S. solfataricus [23]. We have put forward a model specifying that either trimeric SsoIF2 or SsoIF2γ alone binds to the 5′-terminal end of mRNAs and protects them from 5′ to 3′ directional decay during nutrient limitation, for example, when they
Protein and RNA preparation
SsoIF2γ was prepared as described previously [6]. Amino acid replacements and deletions in SsoIF2γ were performed by means of site-directed mutagenesis of a plasmid borne copy of the SsoIF2γ gene using the QuikChange method (Stratagene). The mutant proteins were prepared in the same way as the wild-type protein. Expression and purification of the full-length α-subunit (SsoIF2α) and of its domain 3 (SsoIF2αD3) were performed similarly as described by Stolboushkina et al. [9].
For purification of
Acknowledgements
We thank Drs. S. E. Permyakov and A. S. Kazakov for the help in SPR data collection. The research was supported by the Russian Academy of Sciences, the Russian Foundation for Basic Research (14-04-00406-а and 14-04-31030-mola), the Program of RAS on Molecular and Cellular Biology and the European Molecular Biology Organization short-term fellowship to V.A. (EMBO ASTF 325-2013). The work in U.B. laboratory was supported by grant P21560 from the Austrian Science Fund.
References (39)
- et al.
X-ray structure of translation initiation factor eIF2g: Implications for tRNA and eIF2a binding
J. Biol. Chem.
(2004) - et al.
Structural switch of the γ subunit in an archaeal aIF2αγ heterodimer
Structure
(2006) - et al.
New insights into the interactions of the translation initiation factor 2 from archaea with guanine nucleotides and initiator tRNA
J. Mol. Biol.
(2007) - et al.
Crystal structure of the intact translation initiation factor 2 demonstrates very high conformational flexibility in the α- and β-subunits
J. Mol. Biol.
(2008) - et al.
Crystal structure of the archaeal translation initiation factor 2 in complex with a GTP analogue and Met-tRNAf(Met.)
J. Mol. Biol.
(2013) - et al.
Characterization of GTP-dependent Met-tRNAf binding protein
J. Biol. Chem.
(1977) - et al.
Binding of ATP and messenger RNA by the beta-subunit of eukaryotic initiation factor 2
Biochimie
(1992) - et al.
Classification and evolution of P-loop GTPases and related ATPases
J. Mol. Biol.
(2002) - et al.
Kinetic studies of RNA–protein interactions using surface plasmon resonance
Methods
(2002) - et al.
The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors
EMBO J.
(2002)
Structure of archaeal translational initiation factor 2bg-GDP reveals significant conformational change of the b-subunit and switch 1 region
Proc. Natl. Acad. Sci. U. S. A.
Structure of an archaeal heterotrimeric initiation factor 2 reveals a nucleotide state between GTP and GDP states
Proc. Natl. Acad. Sci. U. S. A.
Conformational transitions in the γ-subunit of the archaeal translation initiation factor 2
Acta Crystallogr., Sect. D: Biol. Crystallogr.
Identification of a second GTP-bound magnesium ion in archaeal initiation factor 2
Nucleic Acids Res.
Translation initiation complex formation in the crenarchaeon Sulfolobus solfataricus
RNA
Translation initiation factor a/eIF2(-γ) counteracts 5′- to 3′-mRNA decay in the archaeon Sulfolobus solfataricus
Proc. Natl. Acad. Sci. U. S. A.
Specific binding of messenger RNA and methionyl-tRNAfMet by the same initiation factor for eukaryotic protein synthesis
Proc. Natl. Acad. Sci. U. S. A.
Structure of the ribosomal protein L1-mRNA complex at 2.1 Å resolution: Common features of crystal packing of L1-RNA complexes
Acta Crystallogr., Sect. D: Biol. Crystallogr.
Ribosomal protein L1 recognizes the same specific structural motif in its target sites on the autoregulatory mRNA and 23S rRNA
Nucleic Acids Res.
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