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1. Synergy and allostery in ligand binding by HIV-1 Nef

Author

Stefan T. Arold, Audrey Restouin, Yves Collette, Sandrine Opi, Safia Aljedani, Christian Dumas, Mingdong Huang, Luc Ponchon, Xavier Morelli, Umar F. Shahul Hameed, Afaque Ahmad Imtiyaz Momin, Adrien Lugari, Xiaoli Shi, Abdullah Aldehaiman, Luyao Wang, Centre de Recherche en Cancérologie de Marseille (CRCM), Aix Marseille Université (AMU)-Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Institut Paoli-Calmettes, Fédération nationale des Centres de lutte contre le Cancer (FNCLCC), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut Paoli-Calmettes, and Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Fédération nationale des Centres de lutte contre le Cancer (FNCLCC)-Aix Marseille Université (AMU)

Subjects
Protein Conformation, alpha-Helical, Engineering, Nef Protein, viruses, Human immunodeficiency virus (HIV), Gene Expression, Protein Engineering, medicine.disease_cause, Crystallography, X-Ray, Ligands, Proto-Oncogene Proteins c-fyn, Biochemistry, SH3 domain, Heterogeneous-Nuclear Ribonucleoprotein K, 0302 clinical medicine, Structural Biology, Cloning, Molecular, Src family, Research Articles, 0303 health sciences, allostery, Chemistry, 030302 biochemistry & molecular biology, virus diseases, Nuclear Proteins, Ligand (biochemistry), Recombinant Proteins, Host-Pathogen Interactions, Thermodynamics, Allosteric Site, 18-crown-6, Protein Binding, tertiary recognition, Allosteric regulation, Genetic Vectors, Biophysics, Crystallographic data, Library science, Computational biology, Molecular Dynamics Simulation, 03 medical and health sciences, Fetus, medicine, Escherichia coli, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Protein Interaction Domains and Motifs, Amino Acid Sequence, nef Gene Products, Human Immunodeficiency Virus, Molecular Biology, Binding selectivity, 030304 developmental biology, Sequence Homology, Amino Acid, business.industry, selectivity, Computational Biology, Cell Biology, Pathogenicity, Protein Structure, Tertiary, autoinhibition, HIV-1, Protein Conformation, beta-Strand, business, Sequence Alignment, 030217 neurology & neurosurgery
Abstract

The Nef protein of human and simian immunodeficiency viruses (HIV and SIV, respectively) boosts viral pathogenicity through its interactions with host cell proteins. Nef has a folded core domain and large flexible regions, each carrying several protein interaction sites. By combining the polyvalency intrinsic to unstructured regions with the binding selectivity and strength of a 3D folded domain, Nef can bind to many different host cell proteins, perturbing their cellular functions. For example, the combination of a linear proline-rich motif and a hydrophobic core domain surface allows Nef to increase affinity and selectivity for particular Src family SH3 domains. Here we investigated whether the interplay between Nef’s flexible regions and its core domain can allosterically influence ligand selection. We found that the flexible regions can bind back to the core domain in different ways, producing distinct conformational states that alter the SH3 domain selectivity and availability of Nef’s functional motifs. The resulting cross-talk might help synergising certain subsets of ligands while excluding others, promoting functionally coherent Nef-bound protein ensembles. Further, we combined proteomic and bioinformatic analyses to identify human proteins that select SH3 domains in the same way as does Nef. We found that only 2–3% of clones from a whole human fetal library displayed a Nef-like SH3 selectivity. However, in most cases this selectivity appears to be achieved by a canonical linear interaction rather than a Nef-like ‘tertiary’ interaction. This analysis suggests that Nef’s SH3 recognition surface has no (or marginally few) cellular counterparts, validating the Nef tertiary binding surface as a promising unique drug target.

Published
2021
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2. Coexpression and Copurification of RNA-Protein Complexes in Escherichia coli

Author

Margot, El Khouri, Marjorie, Catala, Bili, Seijo, Johana, Chabal, Frédéric, Dardel, Carine, Tisné, and Luc, Ponchon

Subjects
Models, Molecular, Operator Regions, Genetic, Escherichia coli Proteins, RNA-Binding Proteins, Drug Resistance, Microbial, Aptamers, Nucleotide, Chromatography, Affinity, RNA, Bacterial, Capsid, Chloramphenicol, Escherichia coli, Nucleic Acid Conformation, RNA, RNA, Viral, Ampicillin, Computer Simulation, Electrophoresis, Polyacrylamide Gel, Cloning, Molecular, Levivirus, Plasmids
Abstract

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows for efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA-protein coexpression in order to express and purify RNA by affinity in native condition. As a proof of concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

Published
2021

4. Correction: Synergy and allostery in ligand binding by HIV-1 Nef

Author

Adrien Lugari, Umar F. Shahul Hameed, Yves Collette, Mingdong Huang, Audrey Restouin, Luc Ponchon, Abdullah Aldehaiman, Luyao Wang, Xavier Morelli, Stefan T. Arold, Afaque Ahmad Imtiyaz Momin, Sandrine Opi, Christian Dumas, Xiaoli Shi, and Safia Aljedani

Subjects
Chemistry, Allosteric regulation, Biophysics, Human immunodeficiency virus (HIV), medicine, Cell Biology, medicine.disease_cause, Molecular Biology, Biochemistry
Published
2021
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5. Bisubstrate analogues as structural tools to investigate m 6 A methyltransferase active sites

Author

Louis Droogmans, Luc Ponchon, Emmanuelle Braud, Mélanie Etheve-Quelquejeu, Laura Iannazzo, Stephanie Oerum, Marjorie Catala, Pierre Barraud, Franck Brachet, Carine Tisné, Colette Atdjian, Morphogénèse du Cerveau des Vertébrés = Morphogenesis of the vertebrate brain (LBD-E10), Laboratoire de Biologie du Développement (LBD), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Laboratoire de Chimie et de Biochimie Pharmacologiques et Toxicologiques (LCBPT - UMR 8601), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de Microbiologie, Université Libre de Bruxelles Institut de Recherches Microbiologiques J.-M. Wiame, Université Libre de Bruxelles [Bruxelles] (ULB), Optimisation Thérapeutique en Neuropsychopharmacologie (VariaPsy - U1144), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Diderot - Paris 7 (UPD7), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université libre de Bruxelles (ULB), Variabilité de réponse aux Psychotropes (VariaPsy - U1144), and Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)

Subjects
Models, Molecular, Methyltransferase, Stereochemistry, SAM analogue, Molecular Conformation, RNA MTases, Biology, Cofactor, m1A, 03 medical and health sciences, chemistry.chemical_compound, Enzyme Inhibitors -- chemistry, 0302 clinical medicine, Catalytic Domain, Methyltransferases -- antagonists & inhibitors -- chemistry -- metabolism, RlmJ, Transferase, [CHIM]Chemical Sciences, Adenine -- analogs & derivatives -- metabolism, Enzyme Inhibitors, TrmK, bisubstrate analogues, Molecular Biology, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, Adenine, Escherichia coli Proteins, Temperature, Substrate (chemistry), RNA, Isothermal titration calorimetry, m6A, Methyltransferases, Cell Biology, Methylation, Sciences bio-médicales et agricoles, RNA binding, Escherichia coli Proteins -- antagonists & inhibitors -- chemistry -- metabolism, 3. Good health, inhibitor, chemistry, 030220 oncology & carcinogenesis, biology.protein, mA, methyltransferase, Research Paper, Protein Binding, Methyl group
Abstract

RNA methyltransferases (MTases) catalyse the transfer of a methyl group to their RNA substrates using most-often S-adenosyl-L-methionine (SAM) as cofactor. Only few RNA-bound MTases structures are currently available due to the difficulties in crystallising RNA:protein complexes. The lack of complex structures results in poorly understood RNA recognition patterns and methylation reaction mechanisms. On the contrary, many cofactor-bound MTase structures are available, resulting in well-understood protein:cofactor recognition, that can guide the design of bisubstrate analogues that mimic the state at which both the substrate and the cofactor is bound. Such bisubstrate analogues were recently synthesized for proteins monomethylating the N6-atom of adenine (m6A). These proteins include, amongst others, RlmJ in E. coli and METLL3:METT14 and METTL16 in human. As a proof-of-concept, we here test the ability of the bisubstrate analogues to mimic the substrate:cofactor bound state during catalysis by studying their binding to RlmJ using differential scanning fluorimetry, isothermal titration calorimetry and X-ray crystallography. We find that the methylated adenine base binds in the correct pocket, and thus these analogues could potentially be used broadly to study the RNA recognition and catalytic mechanism of m6A MTases. Two bisubstrate analogues bind RlmJ with micro-molar affinity, and could serve as starting scaffolds for inhibitor design against m6A RNA MTases. The same analogues cause changes in the melting temperature of the m1A RNA MTase, TrmK, indicating non-selective protein:compound complex formation. Thus, optimization of these molecular scaffolds for m6A RNA MTase inhibition should aim to increase selectivity, as well as affinity., info:eu-repo/semantics/published

Published
2019
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6. Design of cross-linked RNA/protein complexes for structural studies

Author

Clément Dégut, Ronald Micura, Carine Tisné, Pierre Barraud, Luc Ponchon, Veronika Schwarz, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Leopold Franzens Universität Innsbruck - University of Innsbruck, Expression Génétique Microbienne (EGM (UMR_8261 / FRE_3630)), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), and Institut de biologie physico-chimique (IBPC)

Subjects
0301 basic medicine, Models, Molecular, TRNA modification, Biochemistry, Substrate Specificity, 03 medical and health sciences, Bacterial Proteins, RNA, Transfer, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, tRNA Methyltransferases, Crystallography, 030102 biochemistry & molecular biology, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Thermus thermophilus, RNA, Substrate (chemistry), General Medicine, biology.organism_classification, Stem-loop, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], RNA, Bacterial, 030104 developmental biology, Enzyme, chemistry, Covalent bond, Transfer RNA, Biophysics, Protein Binding
Abstract

Crystallographic studies of RNA/protein complexes are primordial for the understanding of recognition determinants and catalytic mechanisms in the case of enzymes. However, due to the flexibility and propensity to conformational heterogeneity of RNAs, as well as the mostly electrostatic interactions of RNA/protein complexes, they are difficult to crystallize. We present here a method to trap the two interacting partners in a covalent complex, based on a modified reactive RNA allowing the use of the full range of common crystallogenesis tools. We demonstrate the practicability of our approach with the production of a covalent complex of the Thermus thermophilus m1A58 tRNA modification enzyme, and a modified stem loop mimicking the natural substrate of the enzyme.

Published
2019
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7. HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity

Author

Luc Ponchon, Bruno Sargueil, Fatima Benattia, Nathalie Ulryck, Nathalie Chamond, Melissa Ameur, Grégoire de Bisschop, Cibles Thérapeutiques et conception de médicaments (CiTCoM - UMR 8038), Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), and Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], HIV Infections, Biochemistry, Substrate Specificity, DEAD-box RNA Helicases, 03 medical and health sciences, RNA specificity, Transcription (biology), ATPase, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Guide RNA, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], ComputingMilieux_MISCELLANEOUS, Ribonucleoprotein, chemistry.chemical_classification, 030102 biochemistry & molecular biology, biology, RNA, Helicase, General Medicine, Biochemical Activity, Cell biology, Kinetics, 030104 developmental biology, Enzyme, chemistry, Cytoplasm, biological substrate, biology.protein, HIV-1, DDX3X, Protein Binding, RNA, Guide, Kinetoplastida
Abstract

International audience; DEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N-and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions.

Published
2018
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8. Backbone resonance assignment of the human uracil DNA glycosylase-2

Author

Hesna Kara, Luc Ponchon, Serge Bouaziz, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Unité de Pharmacologie Chimique et Génétique (UPCG - UMR_S 640/UMR 8151), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC), Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut de Recherche pour le Développement (IRD)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), and Bouaziz, Serge

Subjects
0301 basic medicine, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], viruses, Biology, Vpr, Biochemistry, 03 medical and health sciences, chemistry.chemical_compound, 0302 clinical medicine, Viral life cycle, Structural Biology, Transcription (biology), UNG2, Humans, Uracil-DNA Glycosidase, Nuclear Magnetic Resonance, Biomolecular, Base excision repair, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biochemical phenomena, metabolism, and nutrition, Long terminal repeat, Reverse transcriptase, 3. Good health, 030104 developmental biology, chemistry, Uracil-DNA glycosylase, Host cell cytoplasm, HIV-1, 030217 neurology & neurosurgery, DNA
Abstract

International audience; The HIV-1 viral protein R (Vpr) is incorporated into virus particle during budding suggesting that its presence in the mature virion is required in the early steps of the virus life cycle in newly infected cells. Vpr is released into the host cell cytoplasm to participate to the translocation of the preintegration complex (PIC) into the nucleus for integration of the viral DNA into the host genome. Actually, Vpr plays a key role in the activation of the transcription of the HIV-1 long terminal repeat (LTR), mediates cell cycle arrest in G2 to M transition, facilitates apoptosis and controls the fidelity of reverse transcription. Moreover, Vpr drives the repair enzyme uracil DNA glycosylase (UNG2) towards degradation. UNG2 has a major role in "Base excision repair" (BER) whose main function is to maintain genome integrity by controlling DNA uracilation. The interaction of Vpr with the cellular protein UNG2 is a key event in various stages of retroviral replication and its role remains to be defined. We have performed the structural study of UNG2 by NMR and we report its (1HN, 15N, 13Cα, 13Cβ and 13C') chemical shift backbone assignment and its secondary structure in solution as predicted by TALOS-N. We aim to determine with accuracy by NMR, the residues of UNG2 interacting with Vpr, characterize their interaction and use the local structure of UNG2 and its interface with Vpr to propose potential ligands disturbing this interaction.

Published
2018
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9. The m1A58 modification in eubacterial tRNA: An overview of tRNA recognition and mechanism of catalysis by TrmI

Author

Pierre Barraud, Carine Tisné, Clément Dégut, Luc Ponchon, and Marcia Folly-Klan

Subjects
0301 basic medicine, Stereochemistry, Biophysics, Crystallography, X-Ray, Biochemistry, Catalysis, 03 medical and health sciences, Residue (chemistry), chemistry.chemical_compound, RNA, Transfer, Tetramer, Ribose, Transferase, chemistry.chemical_classification, biology, Thermus thermophilus, Organic Chemistry, Active site, biology.organism_classification, Enzymes, 030104 developmental biology, Enzyme, chemistry, Transfer RNA, biology.protein, Crystallization
Abstract

The enzymes of the TrmI family catalyze the formation of the m(1)A58 modification in tRNA. We previously solved the crystal structure of the Thermus thermophilus enzyme and conducted a biophysical study to characterize the interaction between TrmI and tRNA. TrmI enzymes are active as a tetramer and up to two tRNAs can bind to TrmI simultaneously. In this paper, we present the structures of two TrmI mutants (D170A and Y78A). These residues are conserved in the active site of TrmIs and their mutations result in a dramatic alteration of TrmI activity. Both structures of TrmI mutants revealed the flexibility of the N-terminal domain that is probably important to bind tRNA. The structure of TrmI Y78A catalytic domain is unmodified regarding the binding of the SAM co-factor and the conformation of residues potentially interacting with the substrate adenine. This structure reinforces the previously proposed role of Y78, i.e. stabilize the conformation of the A58 ribose needed to hold the adenosine in the active site. The structure of the D170A mutant shows a flexible active site with one loop occupying in part the place of the co-factor and the second loop moving at the entrance to the active site. This structure and recent data confirms the central role of D170 residue binding the amino moiety of SAM and the exocyclic amino group of adenine. Possible mechanisms for methyl transfer are then discussed.

Published
2016
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10. A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold

Author

Luc Ponchon, Genevieve Beauvais, Frédéric Dardel, Sylvie Nonin-Lecomte, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Neuropsychopharmacologie des addictions. Vulnérabilité et variabilité expérimentale et clinique (NAVVEC - UM 81 (UMR 8206/ U705)), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL (ENSCP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL (ENSCP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Université Paris Diderot - Paris 7 (UPD7), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), and Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Université Paris Diderot - Paris 7 (UPD7)

Subjects
RNase P, Genetic Vectors, Molecular Sequence Data, Ribonuclease H, RNA integrity number, General Biochemistry, Genetics and Molecular Biology, 03 medical and health sciences, Isotopes, RNA, Transfer, Transcription (biology), Gene expression, Escherichia coli, RNase H, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Base Sequence, biology, 030302 biochemistry & molecular biology, RNA, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], RNA, Bacterial, Genetic Techniques, Biochemistry, Transfer RNA, biology.protein, RNA extraction, Chromatography, Liquid, Plasmids
Abstract

RNA production using in vivo transcription by Escherichia coli allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. We describe here a generic protocol for the overproduction and purification of recombinant RNA using liquid chromatography. The strategy utilizes a transfer RNA (tRNA) as a scaffold that can be removed from the RNA of interest by digestion of the fusion RNA at a designed site by RNase H. The tRNA scaffold serves to enhance the stability and to promote the proper expression of its fusion partners. This protocol describes how to construct a tRNA fusion RNA expression vector; to conduct a pilot experiment to assess the yield of the recombinant RNA both before and after processing of the fusion RNA by RNase H; and to purify the target RNA on a large scale for structural or functional studies. This protocol greatly facilitates production of RNA in a time frame of approximately 3 weeks from design to purification. As compared with in vitro methods (transcription, chemical synthesis), this approach is simple, cheap and well suited for large-scale expression and isotope labeling.

Published
2009
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12. Expression and Purification of RNA-Protein Complexes in Escherichia coli

Author

Margueritte, El Khouri, Marjorie, Catala, Bili, Seijo, Johana, Chabal, Carine, Tisné, Frédéric, Dardel, and Luc, Ponchon

Subjects
RNA, Transfer, Escherichia coli, Gene Expression, Nucleic Acid Conformation, Proteins, RNA, Protein Binding
Abstract

For structural, biochemical or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA/protein co-expression in order to express and purify RNA by affinity in native condition. As a proof-of-concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

Published
2015

13. The Endonuclease Domain of Bacteriophage Terminases Belongs to the Resolvase/Integrase/Ribonuclease H Superfamily

Author

Lucienne Letellier, Pascale Boulanger, Gilles Labesse, and Luc Ponchon

Subjects
Genetics, Nuclease, Tn3 transposon, biology, Cell Biology, biology.organism_classification, Biochemistry, Integrase, Bacteriophage, Endonuclease, biology.protein, Ribonuclease, RNase H, Molecular Biology, Bacteriophage T5
Abstract

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.

Published
2006
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14. NMR Solution Structure of Mob1, a Mitotic Exit Network Protein and its Interaction with an NDR Kinase Peptide

Author

Didier Fesquet, Andrey V. Kajava, Christian Dumas, André Padilla, and Luc Ponchon

Subjects
Models, Molecular, Saccharomyces cerevisiae Proteins, Protein Conformation, Molecular Sequence Data, Xenopus, Cell Cycle Proteins, Sequence alignment, Xenopus Proteins, Crystallography, X-Ray, Fungal Proteins, Xenopus laevis, Protein structure, Structural Biology, Animals, Humans, Amino Acid Sequence, Binding site, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, Zinc ion binding, Peptide sequence, Binding Sites, NDR kinase, biology, Phosphoproteins, biology.organism_classification, Zinc, Biochemistry, Helix, Biophysics, Peptides, Sequence Alignment
Abstract

Proteins of the Mob1/phocein family are found in all eukaryotic cells. In yeast, they are activating subunits of Dbf2-related protein kinases involved in cell cycle control. Despite the wide occurrence of these proteins, their biological functions remain poorly understood. Here we report the solution structure of the Mob1 protein from Xenopus laevis solved by heteronuclear multidimensional NMR. The structure reveals a fold constituted by a central left-handed four-helix bundle, one connecting helix, two flanking helices and a long flexible loop. The clustering of two Cys and two His residues, and zinc measurement by atomic absorption spectroscopy support the existence of a zinc ion binding site. Our NMR structure is in good agreement with the recently described X-ray structure of human Mob1-A. Chemical shift perturbations observed upon addition of a peptide encompassing the basic region of the N-terminal regulatory domain of NDR kinase were used to identify and map a specific interaction between Mob1 and this kinase. The chemical shift changes indicate that the main interaction occurs on the acidic and conserved surface of Mob1. This surface was previously hypothesized to be the interaction surface according to the X-ray structure and was identified as functionally important in yeast. Our data suggest that the NDR kinase is a functional Dbf2 homologue in animal cells and contributes to the understanding of the molecular function of Mob1 proteins.

Published
2004
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16. Expression and Purification of RNA–Protein Complexes in Escherichia coli

Author

Margueritte El Khouri, Marjorie Catala, Johana Chabal, Carine Tisné, Bili Seijo, Luc Ponchon, and Frédéric Dardel

Subjects
Expression vector, Chemistry, RNA, Plasma protein binding, medicine.disease_cause, law.invention, Biochemistry, law, Transfer RNA, Gene expression, medicine, Recombinant DNA, RNA extraction, Escherichia coli
Abstract

For structural, biochemical or pharmacological studies, it is required to have pure RNA in large quantities. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. We have extended the "tRNA scaffold" method to RNA/protein co-expression in order to express and purify RNA by affinity in native condition. As a proof-of-concept, we present the expression and the purification of the AtRNA-mala in complex with the MS2 coat protein.

Published
2015
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17. In vivo tmRNA protection by SmpB and pre-ribosome binding conformation in solution

Author

Luc Ponchon, Frédéric Dardel, Bili Seijo, Jean-Michel Saliou, Patrice Vachette, Ehsan Ranaei-Siadat, Sylvie Nonin-Lecomte, Julie Bernauer, Cécile Mérigoux, Sarah Sanglier-Cianférani, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Institut Pluridisciplinaire Hubert Curien (IPHC), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), Algorithms and Models for Integrative Biology (AMIB ), Laboratoire d'informatique de l'École polytechnique [Palaiseau] (LIX), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Laboratoire de Recherche en Informatique (LRI), Université Paris-Sud - Paris 11 (UP11)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Université Paris-Sud - Paris 11 (UP11)-CentraleSupélec-Centre National de la Recherche Scientifique (CNRS)-Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique (CNRS), Laboratoire pour l'Utilisation du Rayonnement Electromagnétique (LURE), Université Paris-Sud - Paris 11 (UP11), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Département Sciences Analytiques et Interactions Ioniques et Biomoléculaires (DSA-IPHC), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS), École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X)-Centre National de la Recherche Scientifique (CNRS)-Laboratoire de Recherche en Informatique (LRI), Laboratoire de Spectrométrie de Masse BioOrganique [Strasbourg] (LSMBO), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), Laboratoire de cristallographie et RMN biologiques ( LCRB - UMR 8015 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Centre National de la Recherche Scientifique ( CNRS ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), ARN-RNP, structure-fonction-maturation ( AREMS ), Université Henri Poincaré - Nancy 1 ( UHP ) -Centre National de la Recherche Scientifique ( CNRS ), Algorithms and Models for Integrative Biology ( AMIB ), Laboratoire d'informatique de l'École polytechnique [Palaiseau] ( LIX ), Centre National de la Recherche Scientifique ( CNRS ) -École polytechnique ( X ) -Centre National de la Recherche Scientifique ( CNRS ) -École polytechnique ( X ) -Laboratoire de Recherche en Informatique ( LRI ), Université Paris-Sud - Paris 11 ( UP11 ) -Institut National de Recherche en Informatique et en Automatique ( Inria ) -CentraleSupélec-Centre National de la Recherche Scientifique ( CNRS ) -Université Paris-Sud - Paris 11 ( UP11 ) -Institut National de Recherche en Informatique et en Automatique ( Inria ) -CentraleSupélec-Centre National de la Recherche Scientifique ( CNRS ) -Inria Saclay - Ile de France, Institut National de Recherche en Informatique et en Automatique ( Inria ), Centre National de la Recherche Scientifique ( CNRS ) -École polytechnique ( X ), Institut Pluridisciplinaire Hubert Curien ( IPHC ), Université Louis Pasteur - Strasbourg I-Centre National de la Recherche Scientifique ( CNRS ), Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS), CentraleSupélec-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-CentraleSupélec-Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS)-Inria Saclay - Ile de France, Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Physique Nucléaire et de Physique des Particules du CNRS (IN2P3)-Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Institut Pluridisciplinaire Hubert Curien (IPHC), ARN-RNP, structure-fonction-maturation (AREMS), Université Henri Poincaré - Nancy 1 (UHP)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-École polytechnique (X), and Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Models, Molecular, tmRNA, Magnetic Resonance Spectroscopy, Protein Conformation, Aucun, Electrophoretic Mobility Shift Assay, Biology, Ribosome, Article, trans-translation, Protein structure, X-Ray Diffraction, Escherichia coli, Protein biosynthesis, Nucleic acid structure, Molecular Biology, ComputingMilieux_MISCELLANEOUS, in vivo protection, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], RNA-Binding Proteins, RNA, Translation (biology), SAXS, SmpB, Molecular biology, NMR, RNA, Bacterial, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Protein Biosynthesis, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Transfer RNA, Biophysics, Nucleic Acid Conformation, Erratum, Ribosomes, Trans-translation, Protein Binding, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

TmRNA is an abundant RNA in bacteria with tRNA and mRNA features. It is specialized in trans-translation, a translation rescuing system. We demonstrate that its partner protein SmpB binds the tRNA-like region (TLD) in vivo and chaperones the fold of the TLD-H2 region. We use an original approach combining the observation of tmRNA degradation pathways in a heterologous system, the analysis of the tmRNA digests by MS and NMR, and co-overproduction assays of tmRNA and SmpB. We study the conformation in solution of tmRNA alone or in complex with one SmpB before ribosome binding using SAXS. Our data show that Mg2+ drives compaction of the RNA structure and that, in the absence of Mg2+, SmpB has a similar effect albeit to a lesser extent. Our results show that tmRNA is intrinsically structured in solution with identical topology to that observed on complexes on ribosomes which should facilitate its subsequent recruitment by the 70S ribosome, free or preloaded with one SmpB molecule.

Published
2014
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18. Insights into bacteriophage T5 structure from analysis of its morphogenesis genes and protein components

Author

Luc Ponchon, Alexis Huet, Madalena Renouard, Cécile Breyton, Rudi Lurz, Pascale Boulanger, Fabrice Confalonieri, Alan R. Davidson, Mohamed Chami, Yvan Zivanovic, Ali Flayhan, Paulette Decottignies, Institut de génétique et microbiologie [Orsay] (IGM), Université Paris-Sud - Paris 11 (UP11)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Centre d'études et de recherche en informatique et communications (CEDRIC), Ecole Nationale Supérieure d'Informatique pour l'Industrie et l'Entreprise (ENSIIE)-Conservatoire National des Arts et Métiers [CNAM] (CNAM), Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Laboratoire pour l'utilisation du rayonnement électromagnétique (LURE), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-MENRT-Centre National de la Recherche Scientifique (CNRS), Muséum national d'Histoire naturelle (MNHN), Institut de biochimie et biophysique moléculaire et cellulaire (IBBMC), Laboratoire de Réactivité de Surface (LRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM)-HESAM Université - Communauté d'universités et d'établissements Hautes écoles Sorbonne Arts et métiers université (HESAM), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), Institut de biologie structurale (IBS - UMR 5075), Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC), Institut de génétique et microbiologie [Orsay] ( IGM ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre National de la Recherche Scientifique ( CNRS ), Laboratoire de cristallographie et RMN biologiques ( LCRB - UMR 8015 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Centre National de la Recherche Scientifique ( CNRS ), Centre d'étude et de recherche en informatique et communications ( CEDRIC ), Ecole Nationale Supérieure d'Informatique pour l'Industrie et l'Entreprise ( ENSIIE ) -Conservatoire National des Arts et Métiers [CNAM] ( CNAM ), Institut de biologie structurale ( IBS - UMR 5075 ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Université Grenoble Alpes ( UGA ) -Université Joseph Fourier - Grenoble 1 ( UJF ), Laboratoire pour l'utilisation du rayonnement électromagnétique ( LURE ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -MENRT-Centre National de la Recherche Scientifique ( CNRS ), Muséum National d'Histoire Naturelle ( MNHN ), Institut de biochimie et biophysique moléculaire et cellulaire ( IBBMC ), Laboratoire de Réactivité de Surface ( LRS ), and Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Centre National de la Recherche Scientifique ( CNRS )

Subjects
MESH : Escherichia coli, MESH : Molecular Sequence Data, viruses, MESH: Amino Acid Sequence, Siphoviridae, Genome, chemistry.chemical_compound, MESH: Capsid, Bacteriophages, MESH : Viral Proteins, Bacteriophage T5, MESH : Siphoviridae, 0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, MESH : Amino Acid Sequence, MESH : Sequence Alignment, 030302 biochemistry & molecular biology, MESH : Bacteriophages, Cell biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Capsid, Lytic cycle, MESH : Capsid, Molecular Sequence Data, Immunology, MESH: Sequence Alignment, MESH: Microscopy, Electron, Microbiology, Viral Proteins, 03 medical and health sciences, Virology, Escherichia coli, MESH: Siphoviridae, Amino Acid Sequence, MESH: Bacteriophages, Gene, 030304 developmental biology, MESH: Molecular Sequence Data, Structure and Assembly, biology.organism_classification, Molecular biology, MESH: Viral Proteins, MESH : Microscopy, Electron, Microscopy, Electron, chemistry, Cytoplasm, Insect Science, Sequence Alignment, DNA, [ SDV.BBM.BS ] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM]
Abstract

Bacteriophage T5 represents a large family of lytic Siphoviridae infecting Gram-negative bacteria. The low-resolution structure of T5 showed the T=13 geometry of the capsid and the unusual trimeric organization of the tail tube, and the assembly pathway of the capsid was established. Although major structural proteins of T5 have been identified in these studies, most of the genes encoding the morphogenesis proteins remained to be identified. Here, we combine a proteomic analysis of T5 particles with a bioinformatic study and electron microscopic immunolocalization to assign function to the genes encoding the structural proteins, the packaging proteins, and other nonstructural components required for T5 assembly. A head maturation protease that likely accounts for the cleavage of the different capsid proteins is identified. Two other proteins involved in capsid maturation add originality to the T5 capsid assembly mechanism: the single head-to-tail joining protein, which closes the T5 capsid after DNA packaging, and the nicking endonuclease responsible for the single-strand interruptions in the T5 genome. We localize most of the tail proteins that were hitherto uncharacterized and provide a detailed description of the tail tip composition. Our findings highlight novel variations of viral assembly strategies and of virion particle architecture. They further recommend T5 for exploring phage structure and assembly and for deciphering conformational rearrangements that accompany DNA transfer from the capsid to the host cytoplasm.

Published
2014
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19. Selective RNase H Cleavage of Target RNAs from a tRNA Scaffold

Author

Genevieve Beauvais, Frédéric Dardel, Sylvie Nonin-Lecomte, Luc Ponchon, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Neuropsychopharmacologie des addictions. Vulnérabilité et variabilité expérimentale et clinique (NAVVEC - UM 81 (UMR 8206/ U705)), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Institut des sciences du Médicament -Toxicologie - Chimie - Environnement (IFR71), Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris - Chimie ParisTech-PSL (ENSCP), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Université Paris Diderot - Paris 7 (UPD7), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5), and Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL (ENSCP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Ecole Nationale Supérieure de Chimie de Paris- Chimie ParisTech-PSL (ENSCP)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD)-Université Paris Descartes - Paris 5 (UPD5)-Université Paris Diderot - Paris 7 (UPD7)

Subjects
0303 health sciences, biology, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Oligonucleotide, RNase P, Chemistry, 030302 biochemistry & molecular biology, RNA, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, RNase PH, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], 03 medical and health sciences, RNase MRP, Biochemistry, Transfer RNA, biology.protein, RNA Cleavage, RNase H, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology
Abstract

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cleavage, we show that the RNA of interest can be isolated in a one-step purification. This method has, in particular, applications in structural investigations of RNA.

Published
2013
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20. Purification of RNA expressed in vivo inserted in a tRNA scaffold

Author

Luc, Ponchon and Frédéric, Dardel

Subjects
Hepatitis B virus, Viral Proteins, RNA, Transfer, Recombinant Fusion Proteins, Escherichia coli, Gene Expression, Humans, Genetic Engineering, Chromatography, Liquid
Abstract

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. In vitro transcription or chemical synthesis are the principal methods to produce RNA. Here, we describe an alternative method allowing RNA production in bacteria and its purification by liquid chromatography. In a few days, between 10 and 100 mg of pure RNA are obtained with this technique.

Published
2012

21. Selective RNase H cleavage of target RNAs from a tRNA scaffold

Author

Luc, Ponchon, Geneviève, Beauvais, Sylvie, Nonin-Lecomte, and Frédéric, Dardel

Subjects
RNA Cleavage, RNA, Transfer, Staining and Labeling, RNA, Ribosomal, 16S, Ribonuclease H, Electrophoresis, Polyacrylamide Gel
Abstract

In vivo overproduction of tRNA chimeras yields an RNA insert within a tRNA scaffold. For some applications, it may be necessary to discard the scaffold. Here we present a protocol for selective cleavage of the RNA of interest from the tRNA scaffold, using RNase H and two DNA oligonucleotides. After cleavage, we show that the RNA of interest can be isolated in a one-step purification. This method has, in particular, applications in structural investigations of RNA.

Published
2012

22. Purification of RNA Expressed In Vivo Inserted in a tRNA Scaffold

Author

Frédéric Dardel and Luc Ponchon

Subjects
Scaffold, biology, Biochemistry, Chemistry, In vivo, Transfer RNA, Gene expression, RNA, In vitro transcription, biology.organism_classification, Chemical synthesis, Molecular biology, Bacteria
Abstract

For structural, biochemical, or pharmacological studies, it is required to have pure RNA in large quantities. In vitro transcription or chemical synthesis are the principal methods to produce RNA. Here, we describe an alternative method allowing RNA production in bacteria and its purification by liquid chromatography. In a few days, between 10 and 100 mg of pure RNA are obtained with this technique.

Published
2012
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23. Large scale expression and purification of recombinant RNA in Escherichia coli

Author

Frédéric Dardel and Luc Ponchon

Subjects
Genetics, Riboswitch, biology, Transcription, Genetic, 5.8S ribosomal RNA, Genetic Vectors, Ribozyme, DNA, Recombinant, RNA, Computational biology, General Biochemistry, Genetics and Molecular Biology, Small hairpin RNA, RNA editing, Transcription (biology), Gene expression, biology.protein, Escherichia coli, Cloning, Molecular, Promoter Regions, Genetic, Molecular Biology, Chromatography, Liquid
Abstract

Stable, folded RNA are involved in many key cellular processes and can be used as tools for biological, pharmacological and/or molecular design studies. However, their widespread use has been somewhat limited by their fragile nature and by the difficulties associated with their production on a large scale, which were limited to in vitro methods. This work reviews the novel techniques recently developed that allow efficient expression of recombinant RNA in vivo in Escherichia coli. Based on the extensive data available on the genetic and metabolic mechanisms of this model organism, conditions for optimal production can be derived. Combined with a large repertoire of RNA motifs which can be assembled by recombinant DNA techniques, this opens the way to the modular design of RNA molecules with novel properties.

Published
2010

24. Ribosome hijacking: a role for small protein B during trans-translation

Author

Frédéric Dardel, Sylvie Nonin-Lecomte, Brice Felden, Reynald Gillet, Noella Germain‐Amiot, Luc Ponchon, Marc Hallier, Laboratoire de cristallographie et RMN biologiques ( LCRB - UMR 8015 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Centre National de la Recherche Scientifique ( CNRS ), Fonction, structure et inactivation d'ARN bactériens, Université de Rennes 1 ( UR1 ), Université de Rennes ( UNIV-RENNES ) -Université de Rennes ( UNIV-RENNES ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), grants from Region Bretagne (PRIR Grant no. 691 and CRB 2004-1483), Action Concertee Incitative BCMS 136, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), ARN régulateurs bactériens et médecine (BRM), Université de Rennes 1 (UR1), Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), EA2063 - biochimie et biologie moléculaire, Université de Reims Champagne-Ardenne (URCA), Laboratoire de Biochimie Pharmaceutique, Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES), Etude du 'Rnome' bacterien. Identification et Caracterisation d'Acides Ribonucleiques Impliques Dans la Survie et la Virulence', Université de Rennes (UNIV-RENNES)-Université de Rennes (UNIV-RENNES)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Lefevre, Annick, Université de Rennes (UR), Université de Rennes (UR)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Nonin-Lecomte, Sylvie

Subjects
Models, Molecular, MESH : Escherichia coli, Prokaryotic Initiation Factor-1, Protein Conformation, MESH : Nucleic Acid Conformation, MESH: Thermus thermophilus, MESH: Escherichia coli Proteins, RNA, Transfer, Amino Acyl, Biochemistry, Ribosome, trans-translation, MESH: Protein Conformation, Protein structure, MESH : Anticodon, MESH : Protein Interaction Mapping, RNA, Ribosomal, 16S, Protein Interaction Mapping, MESH: Nuclear Magnetic Resonance, Biomolecular, Protein biosynthesis, 30S, MESH: Prokaryotic Initiation Factor-1, MESH : Protein Conformation, Genetics, 0303 health sciences, Alanine, MESH: Codon, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Escherichia coli, Escherichia coli Proteins, MESH : Escherichia coli Proteins, 030302 biochemistry & molecular biology, RNA-Binding Proteins, MESH : Protein Binding, SmpB, MESH: RNA, Ribosomal, 16S, RNA, Bacterial, MESH: Nucleic Acid Conformation, ribosome, Transfer RNA, MESH : RNA, Transfer, Amino Acyl, MESH: RNA, Bacterial, MESH: Models, Molecular, Protein Binding, tmRNA, decoding, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], MESH: Alanine, MESH : Models, Molecular, Scientific Report, MESH : RNA-Binding Proteins, Biology, MESH : Nuclear Magnetic Resonance, Biomolecular, MESH : Thermus thermophilus, 03 medical and health sciences, MESH: RNA, Transfer, Amino Acyl, Anticodon, Escherichia coli, [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, MESH: Anticodon, MESH: Protein Binding, Initiation factor, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Codon, Nuclear Magnetic Resonance, Biomolecular, Molecular Biology, [ SDV.BBM ] Life Sciences [q-bio]/Biochemistry, Molecular Biology, 030304 developmental biology, MESH : Ribosomes, Thermus thermophilus, MESH : Codon, MESH: Protein Interaction Mapping, MESH : RNA, Bacterial, MESH : RNA, Ribosomal, 16S, A-site, MESH: RNA-Binding Proteins, MESH : Prokaryotic Initiation Factor-1, Nucleic Acid Conformation, MESH : Alanine, Ribosomes, MESH: Ribosomes, Trans-translation
Abstract

International audience; Tight recognition of codon-anticodon pairings by the ribosome ensures the accuracy and fidelity of protein synthesis. In eubacteria, translational surveillance and ribosome rescue are performed by the 'tmRNA-SmpB' system (transfer messenger RNA-small protein B). Remarkably, entry and accommodation of aminoacylated-tmRNA into stalled ribosomes occur without a codon-anticodon interaction but in the presence of SmpB. Here, we show that within a stalled ribosome, SmpB interacts with the three universally conserved bases G530, A1492 and A1493 that form the 30S subunit decoding centre, in which canonical codon-anticodon pairing occurs. The footprints at positions A1492 and A1493 of a small decoding centre, as well as on a set of conserved SmpB amino acids, were identified by nuclear magnetic resonance. Mutants at these residues display the same growth defects as for DeltasmpB strains. The SmpB protein has functional and structural similarities with initiation factor 1, and is proposed to be a functional mimic of the pairing between a codon and an anticodon.

Published
2009
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25. Recombinant RNA technology: the tRNA scaffold

Author

Frédéric Dardel, Luc Ponchon, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5) - Centre National de la Recherche Scientifique (CNRS), and Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)

Subjects
Riboswitch, 5.8S ribosomal RNA, Computational biology, Biology, 010402 general chemistry, 01 natural sciences, Biochemistry, MESH: Nucleic Acid Amplification Techniques, 03 medical and health sciences, RNA, Transfer, MESH: RNA, Sense (molecular biology), [SDV.BBM] Life Sciences [q-bio]/Biochemistry, Molecular Biology, Escherichia coli, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, Ligase ribozyme, 030304 developmental biology, 0303 health sciences, MESH: Humans, MESH: Escherichia coli, Ribozyme, RNA, Cell Biology, MESH: RNA, Transfer, Molecular biology, 0104 chemical sciences, MESH: Nucleic Acid Conformation, RNA editing, Transfer RNA, biology.protein, Nucleic Acid Conformation, Nucleic Acid Amplification Techniques, Biotechnology
Abstract

International audience; RNA has emerged as a major player in most cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. So far, this has been a bottleneck, as the only methods for producing such pure RNA have been in vitro syntheses. Here we describe a generic approach for expressing and purifying structured RNA in Escherichia coli, using tools that parallel those available for recombinant proteins. Our system is based on a camouflage strategy, the 'tRNA scaffold', in which the recombinant RNA is disguised as a natural RNA and thus hijacks the host machinery, escaping cellular RNases. This opens the way to large-scale structural and molecular investigations of RNA function.

Published
2007
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26. The endonuclease domain of bacteriophage terminases belongs to the resolvase/integrase/ribonuclease H superfamily: a bioinformatics analysis validated by a functional study on bacteriophage T5

Author

Luc, Ponchon, Pascale, Boulanger, Gilles, Labesse, and Lucienne, Letellier

Subjects
Models, Molecular, Protein Folding, Endodeoxyribonucleases, Escherichia coli Proteins, Molecular Sequence Data, Ribonuclease H, Computational Biology, Amino Acid Sequence, HIV Integrase, Siphoviridae, Sequence Alignment, Protein Structure, Tertiary
Abstract

Bacteriophage terminases are essential molecular motors involved in the encapsidation of viral DNA. They are hetero-multimers whose large subunit encodes both ATPase and endonuclease activities. Although the ATPase domain is well characterized from sequence and functional analysis, the C-terminal region remains poorly defined. We describe sequence-structure comparisons of the endonuclease region of various bacteriophages that revealed new sequence similarities shared by this region and the Holliday junction resolvase RuvC and to a lesser extent the HIV integrase and the ribonuclease H. Extensive sequence comparison and motif refinement led to a common signature of terminases and resolvases with three conserved acidic residues engaged in catalytic activity. Sequence analyses were validated by in vivo and in vitro functional assays showing that the nuclease activity of the endonuclease domain of bacteriophage T5 terminase was abolished by mutation of any of the three predicted catalytic aspartates. Overall, these data suggest that the endonuclease domains of terminases operate autonomously and that they adopt a fold similar to that of resolvases and share the same divalent cation-dependent enzymatic mechanism.

Published
2005

27. Encapsidation and transfer of phage DNA into host cells: from in vivo to single particles studies

Author

Lucienne Letellier, Luc Ponchon, Stéphanie Mangenot, and Pascale Boulanger

Subjects
Host (biology), viruses, Phagemid, Virus Assembly, Biophysics, Biological Transport, Biology, Biochemistry, Virology, Genome, chemistry.chemical_compound, Capsid, chemistry, Microscopy, Fluorescence, In vivo, DNA, Viral, Fluorescence microscope, Molecular motor, Bacteriophages, Molecular Biology, DNA
Abstract

A remarkable property of bacteriophages is their capacity to encapsidate large amounts of DNA during morphogenesis and to maintain their genome in the capsid in a very stable form even under extreme conditions. Even as remarkable is the efficiency with which their genome is ejected from the phage particle and transferred into the host bacteria. Biophysical techniques have led to significant progresses in characterizing these mechanisms. The molecular motor of encapsidation of several phages as well as the organization of viral capsids have been described at atomic resolution. Cryo-electron microscopy and fluorescence microscopy have permitted to describe DNA ejection at the level of single phage particles. Theoretical models of encapsidation and ejection have been proposed that can be confronted to experimental data. This review will present the state of the art on the recent advances brought by biophysics in this field. Reference will be given to the work performed on double-stranded DNA phages and on one of its representative, phage T5, our working model.

Published
2005

29. Co-expression of RNA–protein complexes in Escherichia coli and applications to RNA biology

Author

Luc Ponchon, Marjorie Catala, Margueritte El Khouri, Frédéric Dardel, Sylvie Nonin-Lecomte, Carine Tisné, Bili Seijo, Nonin-Lecomte, Sylvie, Laboratoire de cristallographie et RMN biologiques (LCRB - UMR 8015), Université Paris Descartes - Paris 5 (UPD5)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5), Morphogénèse du Cerveau des Vertébrés = Morphogenesis of the vertebrate brain (LBD-E10), Laboratoire de Biologie du Développement (LBD), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), and Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Riboswitch, Viral protein, [SDV.BBM.BS] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Genetic Vectors, Biology, medicine.disease_cause, Methylation, 03 medical and health sciences, Affinity chromatography, Sense (molecular biology), Protein Interaction Mapping, medicine, Genetics, Escherichia coli, RNA Processing, Post-Transcriptional, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Levivirus, chemistry.chemical_classification, 0303 health sciences, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], Base Sequence, 030302 biochemistry & molecular biology, RNA, RNA-Binding Proteins, Molecular biology, Recombinant Proteins, RNA, Bacterial, Enzyme, Biochemistry, chemistry, Transfer RNA, Methods Online, RNA, Transfer, Lys, Capsid Proteins, Plasmids
Abstract

RNA has emerged as a major player in many cellular processes. Understanding these processes at the molecular level requires homogeneous RNA samples for structural, biochemical and pharmacological studies. We previously devised a generic approach that allows efficient in vivo expression of recombinant RNA in Escherichia coli. In this work, we have extended this method to RNA/protein co-expression. We have engineered several plasmids that allow overexpression of RNA-protein complexes in E. coli. We have investigated the potential of these tools in many applications, including the production of nuclease-sensitive RNAs encapsulated in viral protein pseudo-particles, the co-production of non-coding RNAs with chaperone proteins, the incorporation of a post-transcriptional RNA modification by co-production with the appropriate modifying enzyme and finally the production and purification of an RNA-His-tagged protein complex by nickel affinity chromatography. We show that this last application easily provides pure material for crystallographic studies. The new tools we report will pave the way to large-scale structural and molecular investigations of RNA function and interactions with proteins.

Published
2013
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