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Initiation of translation or 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:
aIF2gamma: 1KK0
aIF2alpha-gamma: 2QMU
aIF2: 2QN6

Main conclusions using archaeal aIF2:
Gamma is the central unit bound to the alpha and beta subunits. Gamma 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