DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Ribosome Fidelity and Structure studied by X-ray analysis. – RFS

Ribosome Fidelity and Structure studied by X-ray analysis

The ribosome is a giant ribonucleoprotein cellular assembly that translates genetic code into protein in living cells. The overall goal of our research is to understand how the atomic structure of the ribosome ultimately determines its function. As the ribosome is main target of antibiotics, the study of ribosome is important not only for the fundamental knowledge, but also for developing new therapeutic, which will target the ribosome.

The major goal of the project is to understand the molecular mechanisms of the fidelity of eukaryotic ribosome to the genetic code on atomic level.

Protein synthesis is a tightly regulated cellular process that affects growth, reproduction, and survival in response to both intrinsic and extrinsic cues. Molecular mechanisms responsible for differences in longevity between living organisms are largely unknown, but it was shown that it is related to the fidelity/accuracy of protein translation. Structural investigations of the translation fidelity mechanisms on the atomic level in eukaryote organisms are lacking, because the eukaryotic 80S ribosome is significantly larger and more complex than its bacterial counterpart. Our major goal is to understand the molecular mechanisms of the fidelity of eukaryotic ribosome to the genetic code on atomic level. The mechanisms cannot be understood unless the structures of the participating macromolecules are known at atomic resolution. Thus, we need to crystallize and elucidate the structures of full 80S eukaryotic ribosome with its main functional ligands as messenger RNA, natural cognate/near-cognate transfer RNAs, different translation factors. We will provide the molecular basis of action mode of inhibitors such as aminoglycosides, which alter translation accuracy of eukaryotic ribosome. Presently, we are the only group in the world working on the structure of full 80S eukaryotic ribosome by X-ray analysis. <br />During elongation of protein synthesis maintenance of the reading frame, which is crucial for precise translation of genomic information, is also achieved by the action of the elongation factor eEF2. Interruption in the reading frame is disastrous for a cell and in general can be caused either by direct mutations in eEF2 and impairment in synthesis of the diphthamide residue, a modified histidine, of eEF2 or by imbalance in the protein synthesis apparatus. Thus, our further goal is to solve the crystal structures of the yeast 80S ribosome in the complex with eEF2 in the canonical elongation state and in the state modeling a frameshift event.

X-ray crystallography is an experimental technique of determining the arrangement of atoms within crystals, in which beam of X-ray strikes a crystal and causes the beam of light to spread into many specific directions. Based on the diffraction pattern obtained from X-ray scattering off the periodic assembly of atoms in the crystal, the electron density can be reconstructed. As result of subsequent treatment three-dimensional structure of macromolecule can be produced.
However, the main bottleneck in crystallographic studies is that a well-diffracting crystal must be created, and that the information gleaned about the dynamic nature of the molecules to be studied will be very limited from only a single diffraction experiment. In other words, the price to pay for the high accuracy of X-ray crystallographic structures is that the method is very time-consuming.
The main target of our project is the ribosome, which present the biggest macromolecules in the cell. This ribosome must be crystallized before its structure can be solved by X-Ray crystallography, and for many years there were no ribosome crystals with diffracting abilities. We were the first who solved structure of the bacterial ribosome in its functional state. Preparation of each new type of the crystal (for example ribosome with new drug, other functional ligand) suitable for X-ray crystallography can take several months, sometimes years. A further complication arises since ribosome crystals, as typically seen in RNA crystallography, diffract only poorly which results in electron density maps that are imprecise and difficult to interpret. Therefore special care has to be taken during post-crystallization treatment to avoid damaging the crystals and even for the freezing process itself we could only use the most robust methods.

During first 18 months we accomplished our study of the structural basis for the inhibitory activity of the aminoglycoside during protein synthesis. Aminoglycosides comprise large family of antibiotics, which affect several steps of protein synthesis from translation elongation to termination and ribosome recycling, induce misreading and inhibit inter-subunit rotation of the ribosome. In eukaryotes the effect of aminoglycosides on translation termination made them potent drugs for treatment of human diseases associated with premature termination codons (PTCs). Application aminoglycosides for suppression therapy is limited by their toxicity and low efficiency of the read-through. Many attempts were made to synthesize more potent derivatives. But currently molecular details of action of aminoglycosides in eukaryotes are missing. Therefore, we performed experiments using X-ray crystallography to investigate aminoglycosides interactions with entire 80S eukaryotic ribosome.
Crystal structures for 80S ribosome in complex with paromomycin, gentamicin and TCOO7 (PTCs read-through compound, which combines chemical features of kanamycin and neomycin) were solved at 3.3-3.7Å. We demonstrate the multifaceted mode of action of aminoglycosides on translation in eukaryotes. Structural analysis of aminoglycosides in complex with the eukaryotic 80S ribosome will help in designing new derivatives that are more efficient in PTC read-through and devoid of toxic effects.

We pursued the investigations using dedicated mass spectrometry methods in order to detect and analyze the nucleotide modifications on tRNAs. We focused on tRNAs from Archaea, esp. thermophiles. Indeed, there is no complete set of modified tRNAs in any Archaeum. We have already observed very unusual modifications and also locations for known mutations.

Our objectives will be to obtain bacterial 70S ribosome and eukaryotic 80S ribosome trapped at different stages of reaction of translation in crystal forms, and to solve their structures using X-Ray analysis. By this we can get a snapshots of the protein biosynthesis on atomic level. As a result we will provide new knowledge in understanding of the mechanism of proteins synthesis on the prokaryotic and eukaryotic ribosome in the cell on the atomic level, which can help to explain some processes of regulation of gene expression.
The majority of antibiotics that inhibit translation target the ribosome complex, arresting translation at various stages, spanning initiation, elongation, termination, and recycling.
Comparison of the different drugs binding pockets between bacterial and eukaryotic ribosome on atomic level will give invaluable insight into the development of drugs against viruses, protozoa, fungi and bacteria. Elucidation of the mechanism of biosynthesis of protein on ribosome will bring us the understanding of the molecular mechanism, for example, aging, or disease connected to appearance of aborted (not correct) proteins.

Rozov A et al (2016) Nucleic Acids Res, 44(13):6434-41.
Rozov A et al (2016) Trends in Biochem. Sci, 41(9): 798-814.
Rozov A et al (2016) Nature communications, 7:10457.
Khusainov I et al (2016) Nucleic Acids Res, 44(21): 10491-10504.
Prokhorova I et al (2016) Scientific reports, 6:27720.
Melnikov S et al (2016) J. Mol. Biol., 428:3570-3576.
Maillot J et al (2016) J. Mol. Biol. , 428:2195-2202.
Yusupova G and Yusupov M (2016) Phil.Trans. R. Soc. B 372: 20160184.
Pellegrino S and Yusupova G (2016) Cell Chem Biology, 23, 1319-1321
Westhof, E. (2016) Methods Mol Biol, 1320, 3-8.
Miao Z and Westhof E (2016) Nucleic Acids Res, 44, W562-567.
Grosjean H and Westhof E (2016) Nucleic Acids Res, 44, 8020-8040.
Costa M et al (2016) Science, 354.
Autour A et al (2016) Nucleic Acids Res, 44, 2491-2500.

Protein synthesis is a tightly regulated cellular process that affects growth, reproduction, and survival in response to both intrinsic and extrinsic cues. Translation fidelity assures the production of stable and functional proteomes in prokaryote and eukaryote cells, but it is not an error free biological process. Molecular mechanisms responsible for differences in longevity between living organisms are largely unknown, but it was shown that it is related to the fidelity/accuracy of protein translation. Structural investigations of the translation fidelity mechanisms on the atomic level in eukaryote organisms are lacking, because the eukaryotic 80S ribosome is significantly larger and more complex than its bacterial counterpart. Presently only one technique can conceivably provide the high-resolution structural information the ribosome field of the translation fidelity ultimately requires: X-ray crystallography.
Our major goal is to understand the molecular mechanisms of the fidelity of eukaryotic ribosome to the genetic code on atomic level. The mechanisms cannot be understood unless the structures of the participating macromolecules are known at atomic resolution. Thus, we need to crystallize and elucidate the structures of full 80S eukaryotic ribosome with its main functional ligands, messenger RNA and natural cognate/near-cognate transfer RNAs. More precisely, we need to obtain crystals of full 80S eukaryotic ribosome trapped in the different state of decoding, where the decoding center of eukaryotic ribosome will contain different base pairs at the codon-anticodon helix of messenger RNA (mRNA) and correct or not-correct natural transfer RNAs (tRNA), carrying all natural modifications crucial for the accuracy of genetic information transfer. We will provide the molecular basis of action mode of inhibitors such as aminoglycosides, which alter translation accuracy of eukaryotic ribosome. Recently our group succeeded in solving the first structure of the full eukaryotic 80S ribosome from Saccharomyces cereviseae (Mw 3.4 MDa). Presently, we are the only group in the world working on the structure of full 80S eukaryotic ribosome by X-ray analysis. But only the structure of the vacant eukaryotic ribosome is available. Thus, information of central importance is still missing. We need to visualize the path of the messenger RNA on the eukaryotic ribosome at the atomic level and describe the interactions of all three natural tRNAs with both subunits.
These high-resolution crystal structures will bring first structural insight in the mechanism of ribosome fidelity in the eukaryotes on atomic level. Particularly, these new structures will reveal the features of the eukaryotic translation apparatus that are pivotal for maintaining the mRNA reading frame to achieve efficient and exact translation of genomic information which have been optimized during evolution.
During elongation of protein synthesis maintenance of the reading frame, which is crucial for precise translation of genomic information, is also achieved by the action of the elongation factor eEF2. Interruption in the reading frame (frameshifting) is disastrous for a cell and in general can be caused either by direct mutations in eEF2 and impairment in synthesis of the diphthamide residue, a modified histidine, of eEF2 or by imbalance in the protein synthesis apparatus. Thus, our further goal is to solve the crystal structures of the yeast 80S ribosome in the complex with eEF2 in the canonical elongation state and in the state modeling a frameshift event.
We will also conduct crystallographic studies on the action mode of ribosome inhibitors using prokaryotic and eukaryotic ribosomes as models, and on bacterial ribosome complexes diffracting at high-resolution trapped in different states of decoding.

Project coordination

Gulnara Yusupova (INSTITUT DE GENETIQUE ET DE BIOLOGIE MOLECULAIRE ET CELLULAIRE)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IGBMC INSTITUT DE GENETIQUE ET DE BIOLOGIE MOLECULAIRE ET CELLULAIRE
IBMC - CNRS UPR 9002 CNRS DR ALSACE

Help of the ANR 532,000 euros
Beginning and duration of the scientific project: September 2015 - 48 Months

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