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Portail > Offres > Offre FR550-BRUMIR-009 - Post-doctorat "Etude structurale d'une enzyme de sulfuration [4Fe-4S]-dépendante, impliquée dans la traduction génétique" (H/F)

Post-doc in structural enzymology (M/F)

This offer is available in the following languages:
Français - Anglais

Date Limite Candidature : vendredi 29 octobre 2021

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General information

Reference : FR550-BRUMIR-009
Workplace : PARIS 05
Date of publication : Friday, September 17, 2021
Type of Contract : FTC Scientist
Contract Period : 12 months
Expected date of employment : 1 January 2022
Proportion of work : Full time
Remuneration : Gross salary will vary with candidate's experience starting at 3106€/month before taxes (around 2500€ after taxes) for candidate with less than 2 years post-doctoral experience. See the official CNRS salary table for researcher at http://labexdynamo.ibpc.fr/fp-dynamo-paris/
Desired level of education : PhD
Experience required : Indifferent

Missions

Attention: You must not have resided or carried out your main activity (e.g. work or studies) in France for more than 12 months in the 3 years prior to the recruitment date.
You have research experience in the field relevant to the call.

Structural study of [4Fe-4S]-dependent sulfuration enzymes involved in genetic translation
The use of biochemical techniques and X-ray diffraction will allow us to understand the function of iron-sulfur clusters in catalysis for enzymes involved in the biosynthesis of sulfurated nucleosides.

PI : Béatrice Golinelli-Pimpaneau, Chimie des Processus Biologiques, Collège de France; beatrice.golinelli@college-de-france.fr

Post-transcriptional modifications in tRNA stabilize its tertiary structure, introduce recognition determinants and antideterminants towards RNA-interacting macromolecules and fine-tune the decoding process at the level of both efficiency and fidelity. Moreover, it is becoming increasingly clear that many aspects of RNA metabolism and function are regulated through the dynamic introduction and removal of modifications (1,2). Dynamic tRNA modification regulates cellular response in response to environmental stress and toxicant exposures (3,4,5). Cellular regulation of sulfur-containing tRNAs was found to be tightly coupled and coregulated with translation, in particular upon an increase of temperature (6,7).
We have previously studied biochemically and structurally several sulfuration enzymes that target uridine at positions 34 or 54 in tRNAs (8-11). We have shown that they use a [4Fe-4S] cluster as a cofactor, which led us to propose a new sulfuration mechanism, in which the cluster binds and activates the sulfur atom of the substrate (8).
The structure of E. coli apo-MnmA, which sulfurates U34 in tRNAs, is known for long (12) but we want to obtain its structure with the [4Fe-4S] cluster (holo-MnmA) (9) to definitely establish that it is a [Fe-S]-dependent enzyme.
The project will consist in the crystallization of several holo-MnmA proteins from different bacteria. For some organisms, the highly purified protein is available in high quantity and the work will aim at reconstituting the cluster, purifying the holo-protein and trying to crystallize it. The structure will be solved by molecular replacement.
We want also to understand how sulfur is transferred from L-Cysteine to MnmA. The sulfur atom from free L-cysteine is first mobilized by a PLP-dependent L-cysteine desulfurase. Whereas some organisms like E. coli then use a TusABCDE relay system for sulfur transfer, in some species, the persulfide attached to a specific desulfurase is directly transferred to MnmA. We would like to crystallize such MnmA/desulfurase complexes to gain insight into the sulfur transfer step.

Publications related to the project :
1.Liu, F., Clark, W., Luo, G., Wang, X., Fu, Y., Wei, J., Hao, Z., Dai, Q., Zheng, G., Ma, H. et al. (2016) ALKBH1-Mediated tRNA Demethylation Regulates Translation. Cell, 167, 1897.
2.Roundtree, I.A., Evans, M.E., Pan, T. and He, C. (2017) Dynamic RNA Modifications in Gene Expression Regulation. Cell, 169, 1187-1200.
3.Chan, C.T., Dyavaiah, M., DeMott, M.S., Taghizadeh, K., Dedon, P.C. and Begley, T.J. (2010) A quantitative systems approach reveals dynamic control of tRNA modifications during cellular stress. PLoS Genet, 6, e1001247.
4.Gu, C., Begley, T.J. and Dedon, P.C. (2014) tRNA modifications regulate translation during cellular stress. FEBS Lett, 588, 4287-4296.
5.Huber, S.M., Leonardi, A., Dedon, P.C. and Begley, T.J. (2019) The Versatile Roles of the tRNA Epitranscriptome during Cellular Responses to Toxic Exposures and Environmental Stress. Toxics, 7.
6.Damon, J.R., Pincus, D. and Ploegh, H.L. (2015) tRNA thiolation links translation to stress responses in Saccharomyces cerevisiae. Mol Biol Cell, 26, 270-282.
7.Tyagi, K. and Pedrioli, P.G. (2015) Protein degradation and dynamic tRNA thiolation fine-tune translation at elevated temperatures. Nucleic Acids Res, 43, 4701-4712.
8.Arragain, S., Bimai, O., Legrand, P., Caillat, S., Ravanat, J.L., Touati, N., Binet, L., Atta, M., Fontecave, M. and Golinelli-Pimpaneau, B. (2017) Nonredox thiolation in tRNA occurring via sulfur activation by a [4Fe-4S] cluster. Proc Natl Acad Sci U S A, 114, 7355-7360.
9.hou, J., Lénon, M., Touati, N., Ravanat, J.-L., Velours, C., Fontecave, M., Barras, F. and Golinelli-Pimpaneau, B. (2021) Iron sulfur biology invades tRNA modification: the case of U34 sulfuration. Nucleic Acids Res, 49, 3997-4007.
10. Bimai, O., Legrand, P., Ravanat, J.L., Touati, N., Fontecave, M. and Golinelli-Pimpaneau, B. (2020) The thiolation of uridine 34 in tRNA, which controls protein translation, depends on a [4Fe-4S]-cluster in Methanococcus maripaludis. in preparation.
11. Bimai O, Arragain S, Golinelli-Pimpaneau B. (2020) Structure-based mechanistic insights into catalysis by tRNA thiolation enzymes. Curr Opin Struct Biol. 65, 69-78
12. Numata T, Ikeuchi Y, Fukai S, Suzuki T, Nureki O. (2006) Snapshots of tRNA sulphuration via an adenylated intermediate. Nature. 2006, 442, 419-24

Activities

-Protein purification
-Use of glove boxes (purification, crystallization)
-Chemical reconstitution of iron-sulfur clusters
-Crystallization
-Structures solving and refinement

Skills

Knowledge :
- General knowledge in biochemistry, enzymology and structural biology
- proficiency in scientific English (reading and writting)
- A knowledge of the programs used to solve protein structures (XDS, CCP4, Phenix, COOT) will be advantageous

Know-how :
Strong skills in protein purification, protein crystallization and an interest towards structure determination by X-ray crystallography are required. An experience in working under anaerobic conditions (glove box) is advantageous.

Soft skills
- Innovation, rigor and fiability in performing the work
- Sense of organization.
- Capacity to work in a team

Work Context

The work will be carried out at the Laboratoire de Chimie des Processus Biologiques at the Collège de France (Paris 05), which is equipped for protein purification and crystallization (https://www.college-de-france.fr/site/chimie-processus-biologiques/Plateforme-de-cristallisation-de-proteines.htm). Diffraction data collection takes place at the SOLEIL synchrotron in Saint Aubin (https://www.synchrotron-soleil.fr/fr/lignes-de-lumiere/proxima-1 et https://www.synchrotron-soleil.fr/fr/lignes-de-lumiere/proxima-2a) in remote access or on the site.

The COFUND FP-DYNAMO-PARIS programme offers a unique interdisciplinary environment and a fundamental research initiative in the centre of Paris to train the scientists of tomorrow in the field of physico-chemical biology. The goal of the research programme is to integrate knowledge on gene expression, structural and membrane biology, and bioenergetics in bacteria, chloroplasts and mitochondria in the general context of improving our understanding the biogenesis of energy-transducing membranes. These studies will involve cutting-edge technologies including structural biology (NMR, X-ray crystallography and CryoEM), RNA sequencing, mass-spectrometry, synthetic biology, microfluidics, computational modelling and visualization. Post-doctoral fellows will have the opportunity of developing their research projects in a dynamic scientific environment covering three main research themes: i) RNA biology from bacteria to chloroplasts, ii) Membrane system dynamics and iii) computational modelling of molecular assemblies. They will benefit from the supervision of internationally-recognised experts in their fields, a world-wide network of renowned institutions, cutting-edge research infrastructure and state-of-the-art technical know-how that will allow them to hone their scientific skills, make themselves known and increase their competitiveness on the labour market. Specific training will be proposed to ERs through attendance of the EMBO course on “Laboratory Leadership for Postdocs” included in a retreat in Padua, and involvement in dedicated complementary soft skill training events

Constraints and risks

The contract must start between 1 January 2022 and 31 May.

Additional Information

The application should be written in English and uploaded at https://emploi.cnrs.fr/. It should include a detailed CV, a minimum of two letters of reference, a letter of motivation describing your short and long term career objectives, and a signed declaration stating that you are in compliance with the mobility rule which stipulates that you have not resided or carried out your main activity (work or studies) in France for more than 12 months during the three years preceding the date of recruitment. In addition, the complete file must also be sent to Bruno MIROUX & Magdalena TORTYNA, respectively director and manager of the project at the following email address DYNAMOcofund@ibpc.fr (Tel: 33 1 58 41 50 64). See details on the website http://labexdynamo.ibpc.fr/fp-dynamo-paris/
The position is intended as an initial step in a research career, and the assessment of the applicants will primarily be based on their research qualifications and their potential as researchers with skills that complement and strengthen on-going research within the partner institutions.

Step 1. The applications of each eligible candidate will be evaluated by independent international experts within the relevant research theme. Eligible applicants will be evaluated based on merits, motivation for the program, academic excellence and personal qualifications such as collaborative and communication skills.

Step 2. A maximum of three candidates will be retained for a video interview with the independent experts for each open position (Nov 2021). After the interview, candidates on the short list will be invited to a conference call with the project supervisor and a DYNAMO board member to define the general scope of the project, the training and the research environment. Candidates will be asked to draft a 3-page project proposal, bringing their own original ideas and will be given 2 weeks to submit it.

Step 3. Each project proposal will be evaluated by one of the independent experts and the DYNAMO board member who evaluated the candidate in the previous steps. The final selection will be made based on the scores obtained at steps 1, 2 and 3. The minimum score must be above 20/40. Top candidate will be offered the postdoc position with a starting date from January 1- May 31, 2022.

The CNRS (Centre National de la Recherche Scientifique) is an equal opportunity employer. Furthermore, FP-DYNAMO-PARIS is committed to promoting the role of women in science and thus explicitly encourages women to apply

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