UMR8261 Expression Génétique Microbienne

CNRS / Université Paris Diderot Paris 7

Directeur : Harald Putzer, Directeur-adjoint : Ciarán Condon

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PHYSIOLOGY AND REGULATION OF PROTEIN SYNTHESIS

Principal Investigator : Grégory Boël

 

UMR8261 / CNRS Univ. Paris Diderot Paris 7
Institut de Biologie Physico-Chimique
13, rue P. et M. Curie, 75005 Paris, France
Tel: 01 58 41 51 21

   

   The study of the ribosome and mRNA translation started in the 1960s. Since then, the translation factors, essential to recapitulate the translation process in vitro, have been identified leading to the development of different in vitro translation systems. Since 2000, the technical revolution of structural biology (x-ray crystallography and cryo-electron microscopy) allows us to have a precise view of the molecular mechanisms involved in protein synthesis catalyzed by the ribosome. Nevertheless, even if we can reproduce mRNA translation in vitro under specific conditions (different from the cellular conditions), the current in vitro translation assays are far from matching the efficiency of in vivo translation. This observation suggests that some of the mechanisms and factors controlling the translation process are yet to be discovered

   Our goals are:

1/ To understand at the molecular and physiological level the mechanism of action of newly identified translation factors belonging to the ABC-F protein family.

2/ To identify new translation factors as well as factors that couple translation to other mechanisms.

   Our principal interest is to understand the fundamental aspect of protein synthesis mechanisms and regulations. Furthermore, we try to apply our findings to improve methods for protein synthesis in vivo and in vitro.

   We are currently working with two model organisms:
: Escherichia coli

: Synechocystis sp. PCC 6803

 

 

 

 

 

1) ABC-F translation factors.. 

   The ABC-F family is a family of soluble protein, which belongs to the larger ABC (ATP Binding Cassette) protein superfamily. This superfamily comprises ATPase proteins formed by 2 homologous ABC domains. Most of the ABC proteins are membrane transporters, but the ABC-F family is characterized by soluble proteins. It is the most widespread family of soluble proteins within the ABC superfamily. Four representatives of this ABC-F family are present in Escherichia coli, two in the cyanobacterium Synechocystis, six in Chlamydomonas reinhardtii (including one chloroplastic), five in plants, and three in humans. With our collaborators, we have demonstrated that the most abundant ABC-F family member found in eubacteria, the protein EttA (energy-sensing translational throttle A), is a protein translation factor that gates ribosome entry into the elongation cycle in an ADP-to-ATP ratio-dependent manner.

  In the presence of ADP, EttA inhibits formation of the first peptide bond by the 70S Initiation Complex, thereby blocking entry into the translation elongation cycle. The ATP-bound conformation of EttA stabilizes the 70S Initiation Complex in a conformation favorable to the formation of the first peptide bond. This interaction stimulates ATP hydrolysis by EttA, leading its dissociation from the ribosome, thereby allowing EF-G to catalyze the first round of translocation and drive entry of the 70S ribosomal complex into the elongation cycle. In vivo, cells with the ettA gene knocked out, showed a severe fitness defect in long-term stationary phase, a condition in which the intracellular ATP level is low.

   Since translational apparatus consumes more than half of the total cellular energy and it is thus not unexpected to find a new translation factor that regulates this process. We are currently extending our study of EttA to three other paralogs present in E. coli and ortholog of EttA responsible for antibiotic resistance in several pathogenic bacteria. We also are investigating the function of the two ABC-F proteins present in the cyanobacterium Synechocystis. Our goal is to map the different actions of the ABC-F proteins on the translation apparatus.

 

 

2) Functional characterization of novel translation factors

   In this second task we will develop a screening methodology to identify the phenotype related to translation defects in a mutant collection. This collection will be composed of deletion mutants of proteins known to interact with the ribosome and for which the function remains unclear. In parallel, we will attempt a pull-down approach to identify new factors that interact with stalled ribosomes.

 

 

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The Team

 

Gregory Boël CR1 CNRS
Fares Ousalem Ph.D. student
LABEX Dynamo
Thomas Oïffer

Assistant-Ingénieur, ANR EZOtrad

Anna Liponska Post-Doc, ANR EZOtrad

Alumnus

Amin Omairi-Nasser, Post-doctorant, Labex DYNAMO (01.01.2016 -> 18.04.2017)

 

Publications

  1. Aalberts DP, Boël G, Hunt JF.Codon Clarity or Conundrum? Cell Syst. Jan 4(1):16-19. (2017) doi: 10.1016/j.cels.2017.01.004.

  2. Boël G., Letso R. Neely H., Price W.N., Wong K-H., Su M., Luff J., Valecha M., Everett J., Acton T, Xiao R., Montelione G.T., Aalberts D.P., and Hunt J.F. Codon influence on protein expression in E. coli correlates with mRNA level. Nature 529:358-363 (2016)

  3. Boël G., Smith P.C., Ning W., Englander M.T., Chen B., Hashem Y., Testa A.J., Fischer J.J., Wieden H-J., Frank J., Gonzalez Jr. R.L., Hunt J.F. The ABC-F protein EttA gates ribosome entry into the translation elongation cycle. Nat. Struct. Mol. Bio. 21:143-151 (2014) . News & Views in Nat. Struct. Mol. Bio., 21:15–16

  4. Chen B., Boël G*., Hashem Y*., Ning W., Fei J., Wang W., Gonzalez Jr. R.L., Hunt J.F., Frank J. EttA binds to the ribosomal tRNA exit (E) site and regulates translation by restricting ribosome dynamics. (* Contributed equally) Nat. Struct. Mol. Bio., 21:152-159 (2014)

  5. Pancholi V, Boël G, Jin H. Streptococcus pyogenes Ser/Thr kinase-regulated cell wall hydrolase is a cell division plane-recognizing and chain-forming virulence factor. J Biol Chem., 285(40):30861-30874. (2010)

  6. Mazé A*, Boël G*, Zúñiga M, Bourand A, Loux V, Yebra MJ, Monedero V, Correia K, Jacques N, Beaufils S, Poncet S, Joyet P, Milohanic E, Casarégola S, Auffray Y, Pérez-Martínez G, Gibrat JF, Zagorec M, Francke C, Hartke A, Deutscher J. Complete genome sequence of the probiotic Lactobacillus casei strain BL23. (* Contributed equally) J Bacteriol., 192:2647-2648. (2010)

  7. Boël G., Jin H., and Pancholi V. Inhibition of cell surface export of group A streptococcal anchorless surface dehydrogenase affects bacterial adherence and antiphagocytic properties. Infect. Immun., 73(10):6237-6248 (2005)

  8. Maze A., Boël G., Poncet S., Mijakovic I., Le Breton Y., Benachour A., Monedero V., Deutscher J., Hartke A. The Lactobacillus casei ptsHI47T mutation causes overexpression of a LevR-regulated but RpoN-independent operon encoding a mannose class phosphotransferase system. J Bacteriol., 186:4543-4555. (2004)

  9. Boël G., Pichereau V., Mijakovic I., Mazé A., Poncet S., Gillet S., Giard J.C., Hartke A., Auffray A. and Deutscher J. Is 2-phosphoglycerate-dependent automodification of bacterial enolases implicated in their export? J. Mol. Biol., 337:485-496. (2004)

  10. Mijakovic I., Sandrine P., Boël G., Mazé A., Gillet S., Jamet E., Decottignies P., Grangeasse C., Doublet P., Le Maréchal P. and Deutscher J. Transmembrane modulator-dependent bacterial tyrosine kinase activates UDP-glucose dehydrogenases., EMBO J. 22:4709-4718. (2003)

  11. Boël G., Mijakovic I., Mazé A., Poncet S., Taha M.K., Larribe M., Galinier A. and Deutscher J. Transcription regulators potentially controlled by HPr kinase/phosphorylase in Gram-negative bacteria. J. Mol. Microbiol. Biotechnol., 5:206-215. (2003)

 

Collaborations

Harald Putzer, IBPC
John F. Hunt, Columbia University, New York
Daniel P. Aalbert, Williams college, Williamstown, MA

 

Funding

 

 

 

 

 

 

 

 

 

 

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