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2014

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Thermodynamic/kinetic studies of the translation machinery in action

Research unit

UPR 9002 - Architecture et réactivité de l'ARN (IBMC)
15, rue Rene Descartes 67084 - Strasbourg Cedex

Group

Name: Biophysique et Biologie Structurale

Group leader: DUMAS Philippe - p.dumas@ibmc-cnrs.unistra.fr

Group leader's phone: 03 88 41 70 02

Website: Visit website

Group organization:
- Chercheurs: 4
- ITA: 2
- Doctorants: 2
- Post-Docs: 0
- Autres: 2

Publications of the team linked to the topic (3 last years):
1) Bec G, Meyer B, Gerard MA, Steger J, Fauster K, Wolff P, Burnouf D, Micura R, Dumas P & Ennifar E (2013). Thermodynamics of HIV-1 Reverse Transcriptase in action elucidates the mechanism of action for non-nucleoside inhibitors. J Am Chem Soc., 135(26):9743-52.
2) Burnouf D, Ennifar E, Guedich S, Puffer B, Hoffmann G, Bec G, Disdier F, Baltzinger M & Dumas P (2012). kinITC: a new method for obtaining joint thermodynamic and kinetic data by Isothermal Titration Calorimetry. J Am Chem Soc. 134(1):559-65.
3) Ennifar E, Aslam MW, Strasser P, Hoffmann G, Dumas P, van Delft F (2013). Structure-guided Discovery of a Novel Aminoglycoside Conjugate Targeting HIV-1 RNA Viral Genome. ACS Chemical Biology, 8(11):2509-17.

About PhD

PhD Director: ENNIFAR Eric - e.ennifar@ibmc-cnrs.unistra.fr

Phone: 0388417001

Junior advisor: GUILLAUME Bec

Co-tutely: non

Co-Director: DUMAS Philippe
University of Co-Director: CNRS UPR 9002

About PhD topic :

Title: Thermodynamic/kinetic studies of the translation machinery in action

Project: In bacteria, the translation initiation process involves the binding of the messenger RNA start site and the fMet-tRNAfMet to the 30S ribosomal subunit. The interaction is under control of the initiation factors IF1, IF2 and IF3, resulting in the formation of the 30S initiation complex. The 50S ribosomal subunit then binds to this initiation complex and, after ejection of IFs, leads to the transition to the elongation phase. Detailed structural views of ribosome structures were provided in the past 10 years by many crystal and cryo-EM structures, but thermodynamic data are sparse
Using a Microcal ITC200, the most sensitive isothermal titration calorimeter available, the goal of this project is to collect thermodynamic and, if possible, kinetic data, on the translation elongation and termination using isothermal titration calorimetry (ITC). ITC is a true in-solution technique and is considered the “gold standard” assay for binding since it directly provides, in one single experiment, the complete binding profile between two molecules, without any labelling requirement. In addition, our group recently developed a new ITC approach in order to study successive chemical reactions step by step in one ITC experiment. This strategy, termed incremental ITC (iITC) was successfully used in order to study the polymerization the HIV-1 reverse transcriptase (RT) and the mechanism of action of RT inhibitors. Furthermore, we also developed a new method of ITC data processing, which we named kinITC (kinetic ITC), yielding a complete description of all kinetic parameters, in addition to thermodynamic data (Burnouf, Ennifar et al, JACS 2012). Using incremental ITC, we already obtained the thermodynamic landscape of the translation initiation complex assembly. Our goal is now to follow elongation and termination by successive additions of the required ribosomal factors in the ITC cell. This will first require purification of high amount of each ribosomal factor (tRNA, 30S and 50S subunits, mRNA and ribosomal elongation and termination factors) which will be performed in collaboration with the groups of M Yusupov (IGBMC, Illkirch) and P Romby in our laboratory. In conjunction with the recent ribosomal structural database, these results should provide important insights into the molecular forces that dictate and control the translation initiation and elongation.

Wished skills: Basic skills in biochemistry and molecular biology. Knowledge in biophysics would be valuable, without being a prerequisite.

Expertises which will be acquired during the training: Protein expression and purification, RNA purification, classical ITC
microcalorimetry and new ITC methods, data processing