Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease.
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In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.
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Acknowledgements: We would like to thank Sébastien Brûlé and Bruno Baron for their help with AUC and circular dichroism experiments, and Frédéric Fischer for bioinformatical analysis regarding protein HPB8_1270. We thank Diane Soussan for her help in preliminary experiments of this project, Daniel Vinella with help with the ß-galactosidase activity measurement and all the members of the Unité Pathogenèse de Helicobacter. A.T.A. is part of the Pasteur - Paris University (PPU) International PhD Program. This project has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement N° 665807, and from the Institut Carnot Pasteur Microbes & Santé for A.T.A. M.B.L. was a master student of the UTC (Université de Technologie de Compiègne). Support was provided by “Fondation pour la Recherche Médicale” for the grant DBF20161136767 to H.D.R. and the Pasteur-Weizmann Consortium of “The Roles of Noncoding RNAs in Regulation of Microbial Life Styles and Virulence” to H.D.R. B.F.L., X.Y.P. and A.L. are supported by Wellcome Trust Investigator awards (200873/Z/16/Z and 222451/Z/21/Z). The authors would like to thank the DIM 1HEALTH region Ile-de-France for funding the Centrifection project that has allowed the Optima ultracentrifuge investment. We thank the staff of the OPPL at Harwell Campus, UK for preparing vectors, screening expression conditions for the recombinant proteins, and helpful discussions. We thank Katherine Stott for use of the Biophysics Facility, and Dimtri Chirgadze, Steven Hardwick and Lee Cooper for help with the use of the BiocEM CryoEM facility, both at the University of Cambridge, Department of Biochemistry.
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2041-1723
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Wellcome Trust (222451/Z/21/Z)