Sequences outside recognition sets are not neutral for tRNA aminoacylation. Evidence for nonpermissive combinations of nucleotides in the acceptor stem of yeast tRNAPhe.

Phenylalanine identity of yeast tRNAPhe is governed by five nucleotides including residues A73, G20, and the three anticodon nucleotides (Sampson et al., 1989, Science 243, 1363-1366). Analysis of in vitro transcripts derived from yeast tRNAPhe and Escherichia coli tRNAAla bearing these recognition elements shows that phenylalanyl-tRNA synthetase is sensitive to additional nucleotides within the acceptor stem. Insertion of G2-C71 has dramatic negative effects in both tRNA frameworks. These effects become compensated by a second-site mutation, the insertion of the wobble G3-U70 pair, which by itself has no effect on phenylalanylation. From a mechanistic point of view, the G2-C71/G3-U70 combination is not a "classical" recognition element since its antideterminant effect is compensated for by a second-site mutation. This enlarges our understanding of tRNA identity that appears not only to be the outcome of a combination of positive and negative signals forming the so-called recognition/identity set but that is also based on the presence of nonrandom combinations of sequences elsewhere in tRNA. These sequences, we name "permissive elements," are retained by evolution so that they do not hinder aminoacylation. Likely, no nucleotide within a tRNA is of random nature but has been selected so that a tRNA can fulfill all its functions efficiently.journal articleresearch support, non-u.s. gov't1998 May 08importe

Synthetic polyamines stimulate in vitro transcription by T7 RNA polymerase.

The influence of nine synthetic polyamines on in vitro transcription with T7 RNA polymerase has been studied. The compounds used were linear or macrocyclic tetra- and hexaamine, varying in their size, shape and number of protonated groups. Their effect was tested on different types of templates, all presenting the T7 RNA promoter in a double-stranded form followed by sequences encoding short transcripts (25 to 35-mers) either on single- or double-stranded synthetic oligodeoxyribonucleotides. All polyamines used stimulate transcription of both types of templates at levels dependent on their size, shape, protonation degree, and concentration. For each compound, an optimal concentration could be defined; above this concentration, transcription inhibition occurred. Highest stimulation (up to 12-fold) was obtained by the largest cyclic compound called [38]N6C10.comparative studyjournal articleresearch support, non-u.s. gov't1994 Jul 25importe

Tyrosyl-tRNA synthetase: the first crystallization of a human mitochondrial aminoacyl-tRNA synthetase.

Human mitochondrial tyrosyl-tRNA synthetase and a truncated version with its C-terminal S4-like domain deleted were purified and crystallized. Only the truncated version, which is active in tyrosine activation and Escherichia coli tRNA(Tyr) charging, yielded crystals suitable for structure determination. These tetragonal crystals, belonging to space group P4(3)2(1)2, were obtained in the presence of PEG 4000 as a crystallizing agent and diffracted X-rays to 2.7 A resolution. Complete data sets could be collected and led to structure solution by molecular replacement.journal articleresearch support, non-u.s. gov't2007 Apr 012007 03 30importe

Changis-sur-Marne – Les Pétreaux

La 6e phase de fouille (1998) du site situé dans l’emprise de la Carrière Morillon-Corvol de Changis-sur-Marne (Seine-et-Marne) s’est déroulée sur environ 2,6 ha, et, comme à l’accoutumée, en anticipation des travaux d’extraction de la carrière dont le front de taille se déplace perpendiculairement à la Marne, du nord vers le sud-est (fig. 1). On rappellera que l’occupation humaine de Changis-sur-Marne « les Pétreaux » appartient à la longue durée puisque 15 ha de la nappe alluviale déjà foui..

HSFA1a modulates plant heat stress responses and alters the 3D chromatin organization of enhancer-promoter interactions

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts

Quels leviers agroécologiques mobiliser pour la reconception de systèmes durables en brebis laitière ?

Les aléas climatiques, la fluctuation du prix des produits et des matières premières, les enjeux environnementaux remettent en cause la pérennité des exploitations d’élevage et demandent de re concevoir des systèmes d'élevages plus résilients afin d’assurer leur durabilité. L'enjeu est la transformation des pratiques des agriculteurs afin de mener à des systèmes qui répondent à de multiples performances: agronomiques, économiques, environnementales, sociales...Dans ce contexte, le projet CASDAR AUTELO vise à promouvoir des stratégies alimentaires plus autonomes en élevage de brebis laitières.Pour les élevages de ruminants, une plus grande autonomie alimentaire renvoie au concept d'agroécologie qui nous sert de cadre d’analyse de ce travail. La présente étude a été conduite pour explorer les stratégies d’autonomie développées par des éleveurs de brebis laitières dans les deux principaux bassins de production de lait de brebis: Roquefort et Pyrénées-Atlantiques. Nous avons cherché (i) d'une part à décrire ces stratégies en identifiant les leviers agroécologiques mis en place par les éleveurs et (ii) d'autres part à évaluer la multi-performances de ces élevages afin de relier stratégie et impacts de la re conception agroécologique en élevage.Cette étude repose sur la mise en synergie de données issues des suivis d’élevages réalisés selon la méthodologie INOSYS Réseaux d’élevage et sur un travail d'enquêtes auprès des éleveurs suivis.Nous avons identifié quatre stratégies de fonctionnement des systèmes qui s'appuient sur quatre leviers agroécologiques différents : 1-Favoriser l'autonomie alimentaire de l’exploitation,2-Valoriser les prairies naturelles, 3-Limiter le travail du sol, 4-Développer l'agriculture de conservation. Alors que valoriser les prairies naturelles est associé à un usage limité d'intrants chimiques, l'augmentation de l'autonomie alimentaire passe souvent par un renforcement de l'usage des fertilisants, carburants et pesticides. Alors que limiter le travail du sol, le plus souvent pour réduire la charge de travail et réduire l’érosion des sols,nécessite un usage accru des produits phytosanitaires, l'utilisation de davantage de diversités d'espèces et de types de prairies permet de mieux s'en affranchir et de développer l'agriculture de conservation.Ces stratégies ont été évaluées à l’aide d’indicateurs techniques, économiques et environnementaux.Chaque levier présente avantages et inconvénients : l'autonomie alimentaire est une stratégie particulière qui a un coût lié aux intrants nécessaires;selon la situation d'autres stratégies peuvent être plus agroécologiques.En conclusion, nous montrons qu'améliorer la durabilité en élevage implique que les éleveurs redéfinissent leurs objectifs de production animale et l’usage de la diversité des couverts végétaux. Cette étude est un jalon important pour accompagner la transformation des élevages de brebis laitières afin d'améliorer leur durabilité

Aminoacylation properties of pathology-related human mitochondrial tRNA(Lys) variants

In vitro transcription has proven to be a successful tool for preparation of functional RNAs, especially in the tRNA field, in which, despite the absence of post-transcriptional modifications, transcripts are correctly folded and functionally active. Human mitochondrial (mt) tRNA(Lys) deviates from this principle and folds into various inactive conformations, due to the absence of the post-transcriptional modification m(1)A9 which hinders base-pairing with U64 in the native tRNA. Unavailability of a functional transcript is a serious drawback for structure/function investigations as well as in deciphering the molecular mechanisms by which point mutations in the mt tRNA(Lys) gene cause severe human disorders. Here, we show that an engineered in vitro transcribed “pseudo-WT“ tRNA(Lys) variant is efficiently recognized by lysyl-tRNA synthetase and can substitute for the WT tRNA as a valuable reference molecule. This has been exploited in a systematic analysis of the effects on aminoacylation of nine pathology-related mutations described so far. The sole mutation located in a loop of the tRNA secondary structure, A8344G, does not affect aminoacylation efficiency. Out of eight mutations located in helical domains converting canonical Watson–Crick pairs into G–U pairs or C•A mismatches, six have no effect on aminoacylation (A8296G, U8316C, G8342A, U8356C, U8362G, G8363A), and two lead to drastic decreases (5000- to 7000-fold) in lysylation efficiencies (G8313A and G8328A). This screening, allowing for analysis of the primary impact level of all mutations affecting one tRNA under comparable conditions, indicates distinct molecular origins for different disorders

Identity switches between tRNAs aminoacylated by class I glutaminyl- and class II aspartyl-tRNA synthetases.

High-resolution X-ray structures for the tRNA/aminoacyl-tRNA synthetase complexes between Escherichia coli tRNAGln/GlnRS and yeast tRNAAsp/AspRS have been determined. Positive identity nucleotides that direct aminoacylation specificity have been defined in both cases; E. coli tRNAGln identity is governed by 10 elements scattered in the tRNA structure, while specific aminoacylation of yeast tRNAAsp is dependent on 5 positions. Both identity sets are partially overlapping and share 3 nucleotides. Interestingly, the two enzymes belong to two different classes described for aminoacyl-tRNA synthetases. The class I glutaminyl-tRNA synthetase and the class II aspartyl-tRNA synthetase recognize their cognate tRNA from opposite sides. Mutants derived from glutamine and aspartate tRNAs have been created by progressively introducing identity elements from one tRNA into the other one. Glutaminylation and aspartylation assays of the transplanted tRNAs show that identity nucleotides from a tRNA originally aminoacylated by a synthetase from one class are still recognized if they are presented to the enzyme in a structural framework corresponding to a tRNA aminoacylated by a synthetase belonging to the other class. The simple transplantation of the glutamine identity set into tRNAAsp is sufficient to obtain glutaminylatable tRNA, but additional subtle features seem to be important for the complete conversion of tRNAGln in an aspartylatable substrate. This study defines C38 in yeast tRNAAsp as a new identity nucleotide for aspartylation. We show also in this paper that, during the complex formation, aminoacyl-tRNA synthetases are at least partially responsible for conformational changes which involve structural constraints in tRNA molecules.journal articleresearch support, non-u.s. gov'tresearch support, u.s. gov't, p.h.s.1994 Aug 23importe

Towards Understanding Human Mitochondrial Leucine Aminoacylation Identity

International audienceSpecific recognition of tRNAs by aminoacyl-tRNA synthetases is governed by sets of aminoacylation identity elements, well defined for numerous prokaryotic systems and eukaryotic cytosolic systems. Only restricted information is available for aminoacylation of human mito-chondrial tRNAs, despite their particularities linked to the non-classical structures of the tRNAs and their involvement in a growing number of human neurodegenerative disorders linked to mutations in the corresponding tRNA genes. A major difficulty to be overcome is the preparation of active in vitro transcripts enabling a rational mutagenic analysis, as is currently performed for classical tRNAs. Here, structural and aminoacylation properties of in vitro transcribed tRNA Leu(UUR) are presented. Solution probing using a combination of enzymatic and chemical tools revealed only partial folding into an L-shaped structure, with an acceptor branch but with a floppy anticodon branch. Optimization of aminoacylation conditions allowed charging of up to 75% of molecules, showing that, despite its partially relaxed structure, in vitro transcribed tRNA Leu(UUR) is able to adapt to the synthetase. In addition, mutational analysis demonstrates that the discriminator base as well as residue A14 are important leucine identity elements. Thus, human mitochondrial leucylation is dependent on rules similar to those that apply in Escherichia coli. The impact of a subset of pathology-related mutations on amino-acylation and on tRNA structure, has been explored. These variants do not show significant structural rearrangements and either do not affect aminoacylation (mutations T3250C, T3271C, C3303T) or lead to marked effects. Interestingly, two variants with a mutation at the same position (A3243G and A3243T) lead to markedly different losses in aminoacylation efficiencies (tenfold and 300-fold, respectively)