NMR spectroscopic characterization of ribosomal complexes

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Ribosomes are amongst the most complex biological machineries, being responsible for the conversion of genetic information into functional proteins in both prokaryotic and eukaryotic cells. Ribosomes are large (> 2 MDa or more) macromolecular complexes composed of ribosomal RNA (rRNA, 2/3) and ribosomal proteins (1/3). These rRNA-protein complexes are constituted of two domains, a small subunit ensuring the fidelity of decoding, and a large subunit containing the active site of the ribosome. Crystal structures of individual subunits and complete ribosome particles have elucidated the architecture of these sophisticated macromolecular machines and provided us with unprecedented perception of the general molecular and atomic details of protein synthesis. Notably, the structure of the 50S subunit revealed a tunnel 100 Å long and 10-20 Å wide, through which the nascent chains transit during synthesis before exiting the ribosome. Such a conformation for the exit tunnel can accommodate about 30 residues in an extended chain or 60 residues in alpha-helix conformation. Many biophysical and biochemical studies have focused on ribosomal nascent chains and produced different hypotheses and models, but whether nascent chains adopt secondary structure in the tunnel during translation is not clearly understood.Nuclear Magnetic Resonance (NMR) has the unique ability to study both structural and dynamical aspects in biomolecules at the atomic level, in vitro or in vivo. Substantial progress has been made in the past few years for the structural analysis of large protein complexes using magic-angle-spinning (MAS) techniques on sedimented solution samples. Moreover, proton detection techniques for biological solids in combination with fast magic angle spinning allows to significantly increase the sensitivity of the experiment. These developments open the way to high-resolution investigations of complex biological assemblies that are too large for solution-state NMR, making solid-state NMR a complementary technique in structural biology. Within this project, we employ solid-state NMR like techniques to study ribosomal complexes, including nascent chains (RNCs), specifically reconstituted 50S and 70S ribosomal complexes and ribosome associated factors to better understand nascent chain signalling and communication. The proposed work aims at characterizing not only structural details but also dynamical aspects, which are inaccessible to high resolution X-ray crystallography and cryo-EM reconstruction. Since studies of ribosome complexes involved in co-translational processes require homogeneous RNCs preparations, which have not been crystallized to date, solid-state NMR is the method of choice to obtain atomic-resolution data on these complexes.