Probing the relationship between Gram-negative and Gram-positive S1 proteins by sequence analysis

Escherichia coli ribosomal protein S1 is required for the translation initiation of messenger RNAs, in particular when their Shine–Dalgarno sequence is degenerated. Closely related forms of the protein, composed of the same number of domains (six), are found in all Gram-negative bacteria. More distant proteins, generally formed of fewer domains, have been identified, by sequence similarities, in Gram-positive bacteria and are also termed ‘S1 proteins’. However in the absence of functional information, it is generally difficult to ascertain their relationship with Gram-negative S1. In this article, we report the solution structure of the fourth and sixth domains of the E. coli protein S1 and show that it is possible to characterize their β-barrel by a consensus sequence that allows a precise identification of all domains in Gram-negative and Gram-positive S1 proteins. In addition, we show that it is possible to discriminate between five domain types corresponding to the domains 1, 2, 3, 4–5 and 6 of E. coli S1 on the basis of their sequence. This enabled us to identify the nature of the domains present in Gram-positive proteins and, subsequently, to probe the filiations between all forms of S1

Physiologically relevant reconstitution of iron-sulfur cluster biosynthesis uncovers persulfide- processing functions of ferredoxin-2 and frataxin

Iron-sulfur (Fe-S) clusters are essential protein cofactors whose biosynthetic defects lead to severe diseases among which is Friedreich's ataxia caused by impaired expression of frataxin (FXN). Fe-S clusters are biosynthesized on the scaffold protein ISCU, with cysteine desulfurase NFS1 providing sulfur as persulfide and ferredoxin FDX2 supplying electrons, in a process stimulated by FXN but not clearly understood. Here, we report the breakdown of this process, made possible by removing a zinc ion in ISCU that hinders iron insertion and promotes non-physiological Fe-S cluster synthesis from free sulfide in vitro. By binding zinc-free ISCU, iron drives persulfide uptake from NFS1 and allows persulfide reduction into sulfide by FDX2, thereby coordinating sulfide production with its availability to generate Fe-S clusters. FXN stimulates the whole process by accelerating persulfide transfer. We propose that this reconstitution recapitulates physiological conditions which provides a model for Fe-S cluster biosynthesis, clarifies the roles of FDX2 and FXN and may help develop Friedreich's ataxia therapies

Structure and Functional Analysis of the RNA- and Viral Phosphoprotein-Binding Domain of Respiratory Syncytial Virus M2-1 Protein

Respiratory syncytial virus (RSV) protein M2-1 functions as an essential transcriptional cofactor of the viral RNA-dependent RNA polymerase (RdRp) complex by increasing polymerase processivity. M2-1 is a modular RNA binding protein that also interacts with the viral phosphoprotein P, another component of the RdRp complex. These binding properties are related to the core region of M2-1 encompassing residues S58 to K177. Here we report the NMR structure of the RSV M2-158–177 core domain, which is structurally homologous to the C-terminal domain of Ebola virus VP30, a transcription co-factor sharing functional similarity with M2-1. The partial overlap of RNA and P interaction surfaces on M2-158–177, as determined by NMR, rationalizes the previously observed competitive behavior of RNA versus P. Using site-directed mutagenesis, we identified eight residues located on these surfaces that are critical for an efficient transcription activity of the RdRp complex. Single mutations of these residues disrupted specifically either P or RNA binding to M2-1 in vitro. M2-1 recruitment to cytoplasmic inclusion bodies, which are regarded as sites of viral RNA synthesis, was impaired by mutations affecting only binding to P, but not to RNA, suggesting that M2-1 is associated to the holonucleocapsid by interacting with P. These results reveal that RNA and P binding to M2-1 can be uncoupled and that both are critical for the transcriptional antitermination function of M2-1

ETUDE PAR RMN STATIQUE ET DYNAMIQUE EN SOLUTION ET EN PHASE SOLIDE DE COMPLEXES ET DE CLUSTERS COMPORTANT DES NOYAUX QUADRUPOLAIRES. NOUVEAUX DOMAINES D'APPLICATION DE LA RMN HRMAS EN CHIMIE INORGANIQUE

CE TRAVAIL COMPORTE DEUX PARTIES. LA PREMIERE PARTIE A ETE CONSACREE A L'ETUDE D'ECHANGES CHIMIQUES PAR RMN DE NOYAUX QUADRUPOLAIRES. LA DEUXIEME PARTIE EXPLORE DE NOUVELLES POSSIBILITES D'APPLICATION DE LA RMN HRMAS. LA PREMIERE PARTIE MONTRE QUE LES NOYAUX QUADRUPOLAIRES, QUI SONT GENERALEMENT DIFFICILES A OBSERVER, CONSTITUENT NEANMOINS UNE ALTERNATIVE INTERESSANTE PAR RAPPORT AUX NOYAUX DIPOLAIRES POUR APPREHENDER LA DYNAMIQUE ET LES ECHANGES CHIMIQUES EN SOLUTION. LE NOYAU 1 3 3CS A ETE UTILISE POUR ETUDIER L'ECHANGE MULTISITE DU CATION CESIUM LIE A DES LIGANDS CALIX-BIS-COURONNES, QUI SONT DES CANDIDATS POTENTIELS POUR LE TRAITEMENT DE DECHETS RADIOACTIFS. LA DYNAMIQUE DES CHAINES POLYETHERS ET LEUR INFLUENCE SUR LA DECOMPLEXATION DU CATION ONT ETE ANALYSEES. LA RMN MAS 1 3 3CS, UTILISEE JUSQU'A PRESENT SUR DES COMPOSES INORGANIQUES, A ETE APPLIQUEE A DES COMPLEXES CRISTALLISES AFIN D'ACCEDER AUX PARAMETRES QUADRUPOLAIRES. CEUX-CI ONT VALIDE LES PARAMETRES OBTENUS POUR LA MICRODYNAMIQUE. LE NOYAU 5 9CO A PERMIS DE MIEUX CARACTERISER LE COMPORTEMENT FLUXIONNEL DU CLUSTER DE COBALT CO 4(CO) 1 2. C'EST LE SEUL NOYAU PRESENT SUR CE CLUSTER QUI PERMETTE DE QUANTIFIER L'ECHANGE INTRAMOLECULAIRE DES LIGANDS CARBONYLES, SI L'ON TIENT COMPTE DE LA RELAXATION QUADRUPOLAIRE. DANS LA DEUXIEME PARTIE, L'EXEMPLE DE TENSIOACTIFS INCLUS DANS UNE SILICE MESOPOREUSE A MONTRE QUE LA RMN HRMAS (HIGH RESOLUTION MAGIC ANGLE SPINNING) PERMET D'OBTENIR DES SPECTRES 1H DE HAUTE RESOLUTION DANS CES MATERIAUX ET QUE CES ESPECES ONT UN COMPORTEMENT DYNAMIQUE PROCHE DU SOLIDE. LA RMN HRMAS A EGALEMENT ETE TESTEE SUR DES CLUSTERS DE COBALT GREFFES SUR DIFFERENTES RESINES AVEC LES NOYAUX 1H, 1 3C, 3 1P ET 5 9CO. LES RESULTATS OBTENUS MONTRENT QUE LA RMN HRMAS EST UN OUTIL D'INVESTIGATION POTENTIEL POUR DES MOLECULES ORGANOMETALLIQUES ET QU'UN MATERIAU MESOPOREUX PEUT ETRE UTILISE COMME SUPPORT SOLIDE. CECI ELARGIRAIT LE DOMAINE D'APPLICATION D'UNE TECHNIQUE GENERALEMENT UTILISEE EN CHIMIE COMBINATOIRE.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

1H, 13C and 15N resonance assignments of σS activating protein Crl from Salmonella enterica serovar Typhimurium

International audienceThe general stress response in Enterobacteria, like Escherichia coli or Salmonella, is controlled by the transcription factor σ(S), encoded by the rpoS gene, which accumulates during stationary phase growth and associates with the core RNA polymerase enzyme (E) to promote transcription of genes involved in cell survival. Tight regulation of σ(S) is essential to preserve the balance between self-preservation under stress conditions and nutritional competence in the absence of stress. Whereas σ factors are generally inactivated upon interaction with anti-sigma proteins, σ(S) binding by the Crl protein facilitates the formation of the holoenzyme Eσ(S), and therefore σ(S)-controlled transcription. Previously, critical residues in both Crl and σ(S) were identified and assigned to the binding interface in the Crl-σ(S) complex. However, high-resolution structural data are missing to fully understand the molecular mechanisms underlying σ(S) activation by Crl, in particular the possible role of Crl in triggering domain rearrangements in the multi-domain protein σ(S). Here we provide the (1)H, (13)C and (15)N resonance assignments of Salmonella enterica serovar Typhimurium Crl, as a starting point for CrlSTM structure determination and further structural investigation of the CrlSTM-σ STM (S) complex

Intrinsic disorder contribution to the interaction between the potyvirus VPg and the translation initiation factor eIF4E: optimization of the expression of 15N label proteins for NMR studies

Intrinsic disorder contribution to the interaction between the potyvirus VPg and the translation initiation factor eIF4E: optimization of the expression of 15N label proteins for NMR studies. 16. Rencontres de Virologie Végétale (RVV 2017

Unravelling successive steps in Fe−S cluster biosynthesis by the mitochondrial ISC machinery: contributions from NMR focusing on the ISCU/IscU scaffold protein

International audienc

Resonance assignment of the ribosome binding domain of E. coli ribosomal protein S1.

International audienceRibosomal protein S1 is an essential actor for protein synthesis in Escherichia coli. It is involved in mRNA recruitment by the 30S ribosomal subunit and recognition of the correct start codon during translation initiation. E. coli S1 is a modular protein that contains six repeats of an S1 motif, which have distinct functions despite structural homology. Whereas the three central repeats have been shown to be involved in mRNA recognition, the two first repeats that constitute the N-terminal domain of S1 are responsible for binding to the 30S subunit. Here we report the almost complete (1)H, (13)C and (15)N resonance assignment of two fragments of the 30S binding region of S1. The first fragment comprises only the first repeat. The second corresponds to the entire ribosome binding domain. Since S1 is absent from all high resolution X-ray structures of prokaryotic ribosomes, these data provide a first step towards atomic level structural characterization of this domain by NMR. Chemical shift analysis of the first repeat provides evidence for structural divergence from the canonical OB-fold of an S1 motif. In contrast the second domain displays the expected topology for an S1 motif, which rationalizes the functional specialization of the two subdomains

The respiratory syncytial virus non-structural protein 1 and its interaction with the MED25 sub-unit of the mediator complex

National audienc