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Initiation
of translation or
How to select the correct initiation codon on the messenger RNA?
How to select the correct initiation codon on the messenger RNA?
Funded by the CNRS, the Ecole Polytechnique and the Agence Nationale de la Recherche (PCV program, MASTIC project: A Multi-resolution Approach for the Structure of Translation Initiation super-Complexes)
In
all organisms, selection of
the correct start codon requires the
recruitment of a specialized tRNA molecule: the initiator
tRNA, systematically carrying a methionine. Even if this process is universal, in each domain of life,
distinct mechanisms support the selection of the initiator
tRNA. In addition to their mechanistic interest, comparative studies of
translational mechanisms in the three domains of life can also bring
information on the LUCA (Last Universal Common Ancestor) and therefore
on the origin of life.
Initiation is the rate-limiting step of translation. This step of the translational process is critical for the control of many cellular processes, such as Response to amino acids starvation, Development and cellular differentiation, Cancer, ...
Translation initiation involves high order macromolecular complexes
Understanding how networks of interactions between the numerous cellular partners ensure accuracy of the initiation of translation requires functional and structural studies of high-order macromolecular complexes.
Previously, we were mainly involved in the study of the bacterial translation process. In particular, bacterial methionyl-tRNA synthetases as well as the role of the formylation of the initiator tRNAfMet were extensively studied using genetics, biochemistry and structural biology.
Now, we focus our attention on translation initiation in eucaryotes. We also use archaeal systems as simplified models of eucaryotic translation initiation.
Eukaryotic and archaeal translation initiation factor 2: a heterotrimeric tRNA carrier
One part of our recent work deals with the study of the e/aIF2 protein. Eukaryotic/archaeal translation initiation factor 2 (e/aIF2) is a heterorimeric protein resulting from the association of three subunits, alpha, beta and gamma. e/aIF2 binds methionylated initiator tRNA with high affinity (dissociation constant in the nanomolar range) in a GTP-dependent manner and plays a key role in the selection of the correct start codon on messenger RNA. e/aIF2 function requires the hydrolysis of one GTP molecule. In eucaryotes and in archaea, the heterotrimeric e/aIF2 protein carries initiator Met-tRNAiMet towards the ribosome. When the pairing between AUG codon and CAU anticodon is correct, e/aIF2 is released from the ribosome in a GDP-bound state. Therefore, the selection of the start codon is achieved through control of the nucleotide state of e/aIF2.
We have studied initiator tRNA binding to e/aIF2 using a variety of biochemical and structural tools.
Some relevant PDB files:Initiation is the rate-limiting step of translation. This step of the translational process is critical for the control of many cellular processes, such as Response to amino acids starvation, Development and cellular differentiation, Cancer, ...
Translation initiation involves high order macromolecular complexes
Understanding how networks of interactions between the numerous cellular partners ensure accuracy of the initiation of translation requires functional and structural studies of high-order macromolecular complexes.
Previously, we were mainly involved in the study of the bacterial translation process. In particular, bacterial methionyl-tRNA synthetases as well as the role of the formylation of the initiator tRNAfMet were extensively studied using genetics, biochemistry and structural biology.
Now, we focus our attention on translation initiation in eucaryotes. We also use archaeal systems as simplified models of eucaryotic translation initiation.
Eukaryotic and archaeal translation initiation factor 2: a heterotrimeric tRNA carrier
One part of our recent work deals with the study of the e/aIF2 protein. Eukaryotic/archaeal translation initiation factor 2 (e/aIF2) is a heterorimeric protein resulting from the association of three subunits, alpha, beta and gamma. e/aIF2 binds methionylated initiator tRNA with high affinity (dissociation constant in the nanomolar range) in a GTP-dependent manner and plays a key role in the selection of the correct start codon on messenger RNA. e/aIF2 function requires the hydrolysis of one GTP molecule. In eucaryotes and in archaea, the heterotrimeric e/aIF2 protein carries initiator Met-tRNAiMet towards the ribosome. When the pairing between AUG codon and CAU anticodon is correct, e/aIF2 is released from the ribosome in a GDP-bound state. Therefore, the selection of the start codon is achieved through control of the nucleotide state of e/aIF2.
We have studied initiator tRNA binding to e/aIF2 using a variety of biochemical and structural tools.
aIF2gamma: 1KK0
aIF2alpha-gamma: 2QMU
aIF2: 2QN6
Main conclusions using archaeal aIF2:
Gamma is the central unit bound to the alpha and beta subunits. Aamma binds the tRNA molecule; however the binding affinity is very low as compared to that measured with the complete heterotrimer.
Beta has only a small effect on tRNA binding affinity.
Alpha strongly increases the tRNA binding affinity.
Structural studies of the aIF2:GDPNP:Met-tRNAfMet complex
We have determined the 5 Å resolution crystal structure of the ternary complex formed by archaeal aIF2 from Sulfolobus solfataricus, the GTP analog GDPNP and methionylated initiator tRNA. The 3D model is further supported by solution studies using small-angle X-ray scattering. The tRNA is bound by the alpha and gamma subunits of aIF2. Contacts involve the elbow of the tRNA and the minor groove of the acceptor stem, but not the T-stem minor groove. We conclude that despite considerable structural homology between the core gamma subunit of aIF2 and the elongation factor EF1A, these two G proteins of the translation apparatus use very different tRNA-binding strategies.
Highly anisotropic diffraction; 6.3 Å max initially.
Availability of a crystal with low mosaicity + Pilatus detector: 1 dataset to 5 Å resolution with high redundancy (6.5)
PDB file: 3V11
Proxima-1 beamline SOLEIL- French synchrotron facility-collaboration with Andy Thompson
Solution studies by Small Angle X-ray Scattering
SWING beamline SOLEIL- French synchrotron facility-collaboration with Javier Perez.
SAXS data are consistent with a solution structure of the complex identical to that observed in the crystal.
Accurate initiation of translation: Selection of the initiator tRNA by aIF2
Towards a model for the binding of the ternary initiation complex to the small ribosomal subunit
Recent publications related to the "translation initiation" research theme
Naveau M, Lazennec-Schurdevin C, Panvert M, Dubiez E, Mechulam Y, Schmitt E. Roles of yeast eIF2α and eIF2β subunits in the binding of the initiator methionyl-tRNA. Nucleic Acids Res. 2012 Nov 27.
E. Schmitt, M. Panvert, C. Lazennec-Schurdevin, P-D Coureux, J. Perez, A. Thompson and Y. Mechulam (2012). Structure of the ternary initiation complex aIF2–GDPNP–methionylated initiator tRNA. Nature Structural and Molecular Biology, Nat. Struct. Mol. Biol., 9, 450-45
Schmitt E, Naveau M, Mechulam Y. (2010). Eukaryotic and archaeal translation initiation factor 2: a heterotrimeric tRNA carrier. FEBS Lett. 584:405-12. Invited review.
Naveau M, Lazennec-Schurdevin C, Panvert M, Mechulam Y, Schmitt E. (2010). tRNA binding properties of eukaryotic initiation factor 2 from Encephalitozoon cuniculi. Biochemistry, 49:8680-8688.
Barraud P, Schmitt E, Mechulam Y, Dardel F, Tisné C. (2008). A unique conformation of the anticodon stem-loop is associated with the capacity of tRNAfMet to initiate protein synthesis. Nucleic Acids Res.36:4894-901
Mechulam Y, Guillon L, Yatime L, Blanquet S and Schmitt E (2007). Protection-based assays to measure aminoacyl-tRNA binding to translation initiation factors. Methods Enzymol. 430, « Translation Initiation: Reconstituted Systems and Biophysical Methods ». Jon Lorsch ed.. 265-281.
Yatime, L., Mechulam, Y., Blanquet, S. and Schmitt, E. (2007). Structure of an archaeal heterotrimeric initiation factor 2 reveals a novel nucleotide state between the GTP and the GDP ones". PNAS, 104, 18445-18450
Guillon, L., Schmitt, E., Blanquet, S. and Mechulam, Y. (2005). Initiator tRNA binding by e/aIF5B, the eukaryotic/archaeal homologue of bacterial initiation factor IF2. Biochemistry, 44, 15594-15601.
Yatime, L., Mechulam, Y., Blanquet, S. and Schmitt, E. (2006) Structural switch of the g subunit in an archaeal aIF2ag heterodimer. Structure, 14, 119-128.
Yatime, L., Schmitt, E., Blanquet, S. and Mechulam, Y.(2005). Structure-function relationships of the intact aIF2alpha subunit from the archaeon Pyrococcus abyssi Biochemistry.44, 8749-8756.
L. Yatime, E. Schmitt, S. Blanquet, Y.Mechulam. (2004). Functional molecular mapping of archaeal translation initiation factor 2. J. Biol. Chem. 279, 15984-15993.
Schmitt, E., Blanquet, S. & Mechulam, Y. (2002). The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors. EMBO J. 21: 1821-1832
