Structural study of [4Fe-4S]-dependent sulfuration enzyme MnmA involved in genetic translation

PI:  Béatrice Golinelli-Pimpaneau

Chair of Chemistry for Biological Processes at the Collège de France

1-year contract

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). In particular, 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 most likely 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.

A biochemist with experience in protein purification, crystallization, preferentially under anaerobic conditions (glove box), and with some background in protein structure solving by X-ray crystallography is welcome.

  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. Zhou, 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


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