of translation or
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
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, ...
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
synthetases as well as the role of the formylation of the initiator tRNAfMet
were extensively studied using genetics, biochemistry and structural
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:
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
Structural studies of the
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)
Proxima-1 beamline SOLEIL- French synchrotron facility-collaboration
with Andy Thompson
Solution studies by Small Angle
SWING beamline SOLEIL- French synchrotron facility-collaboration with
SAXS data are consistent with
a solution structure of the complex identical to that observed in the
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
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
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.
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
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.
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,
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.
Mechulam, Y., Blanquet, S. and Schmitt, E. (2006) Structural
switch of the g subunit in an archaeal aIF2ag heterodimer. Structure,
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,
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