Research in the last few decades has revealed the great capacity of the RNA molecule. RNA, which previously was assumed to play a main role only as an intermediate in the translation of genes to proteins, is today known to play many important roles in the cell in addition to that as a messenger RNA and transfer RNA, including the ability to catalyze reactions and gene regulations at various levels. This thesis investigates several computational aspects of RNA. We will discuss identification of novel RNAs and RNAs that are known to exist in related species, RNA secondary structure prediction, as well as more general tools for analyzing, visualizing and classifying RNA sequences. We present two benchmark studies concerning RNA identification, both de novo identification/characterization of single RNA sequences and homology search methods. We develope a novel algorithm for analysis of the RNA folding landscape that is based on the nearest neighbor energy model adopted in many secondary structure prediction programs. We implement this algorithm, which computes structural neighbors of a given RNA secondary structure, in the program RNAbor, which is accessible on a web server. Furthermore, we combine a mutual information based structure prediction algorithm with a sequence logo visualization to create a novel visualization tool for analyzing an RNA alignment and identifying covarying sites. Finally, we present extensions to sequence logos for the purpose of tRNA identity analysis. We introduce function logos, which display features that distinguish functional subclasses within a large set of structurally related sequences, as well as the inverse logos, which display underrepresented features. For the purpose of comparing tRNA identity elements between different taxa we introduce two contrasting logos, the information difference and the Kullback-Leibler divergence difference logos.

Antibiotic resistance is a growing problem on a global scale. Increasing numbers of bacteria resistant toward one or multiple antibiotics could return us to the high mortality rates for infectious diseases of the pre-antibiotic era. The need for development of new classes of antibiotics is great as is increased understanding of the mechanisms underlying the development of antibiotic resistance. We have investigated the emergence of resistance to peptide deformylase inhibitors, a new class of antibiotics that target bacterial protein synthesis. The fitness of resistant mutants as well as their propensity to acquire secondary compensatory mutations was assessed in order to gain some insight into the potential clinical risk of resistance development. Most of this work was done in the bacterium Salmonella typhimurium, due to the availability of excellent genetic tools to study these phenomena. In addition, we have studied the bacterium Staphylococcus aureus as peptide deformylase inhibitors have been shown to have the greatest effect on Gram-positive organisms. In the course of this work we also examined the mechanistic aspects of translation initiation. Using a cell-free in vitro translation system we studied the effects of various components on translation initiation. These results have been combined with results obtained from resistant and compensated bacterial strains in vivo to gain new insights into the mechanisms of translation initiation.

Surprisingly little is known about the organization and distribution of tRNAs and tRNA-related sequences on a genome-wide scale. While tRNA complements are usually reported in passing as part of genome annotation efforts, and peculiar features such as the tandem arrangements of tRNAs in Entamoeba histolytica have been described in some detail, comparative studies are rare. We therefore set out to systematically survey the genomic arrangement of tRNAs in a wide range of eukaryotes to identify common patterns and taxon-specific peculiarities. We found that tRNA complements evolve rapidly and that tRNA locations are subject to rapid turnover. At the phylum level, distributions of tRNA numbers are very broad, with standard deviations on the order of the mean. Even within fairly closely related species, we observe dramatic changes in local organization. Consistent with this variability, syntenic conservation of tRNAs is also poor in general, with turn-over rates comparable to those of unconstrained sequence elements. We conclude that the genomic organization of tRNAs shows complex, lineage-specific patterns characterized by extensive variability, and that this variability is in striking contrast to the extreme levels of sequence-conservation of the tRNA genes themselves. Our comprehensive analysis of eukaroyotic tRNA distributions provides a basis for further studies into the interplay between tRNA gene arrangements and genome organization in general. Secondly, we focused on the investigation of small non-coding RNAs (ncRNAs) from whole transcriptome data. Since ncRNAs constitute a significant part of the transcriptome, we explore this data to detect and classify patterns derived from transcriptome-associated loci. We selected three distinct ncRNA classes: microRNAs, snoRNAs and tRNAs, all of which undergo maturation processes that lead to the production of shorter RNAs. After mapping the sequences to the reference genome, specific patterns of short reads were observed. These read patterns appeared to reflect RNA processing and, if so, should specify the RNA transcripts from which they are derived. In order to investigate whether the short read patterns carry information on the particular ncRNA class from which they orginate, we performed a random forest classification on the three distinct ncRNA classes listed above. Then, after exploring the potential classification of general groups of ncRNAs, we focused on the identification of small RNA fragments derived from tRNAs. After mapping transcriptome sequence data to reference genomes, we searched for specific short read patterns reflecting tRNA processing. In this context, we devised a common tRNA coordinate system based on conservation and secondary structure information that allows vector representation of processing products and thus comparison of different tRNAs by anticodon and amino acid. We report patterns of tRNA processing that seem to be conserved across species. Though the mechanisms and functional implications underlying these patterns remain to be clarified, our analysis suggests that each type of tRNA exhibits a specific pattern and thus appears to undergo a characteristic maturation process.

The complete sequence of mitochondrial DNA from Diuraphis noxia was obtained and characterized. The mitogenome contains a standard set of 13 protein-coding genes, 19 tRNA genes, 2 ribosomal RNA genes. A+T-rich and ?repets? regions in the same order as those of the other analyzed aphids. Comparison to mtDNAs from other Sternorrhyncha species obtained from GenBank revealed possible markers for studies on population differentiation. Phylogenetic analysis using parsimony and maximum likelihood confirmed the classification of Diuraphis noxia into the Aphididae.

No description available.

During translation, the transfer RNAs (tRNAs) play the crucial role of adaptors between the messenger RNA and the amino acids. The tRNAs are first transcribed as pre-tRNAs which are then maturated. During this maturation, several nucleosides are modified by tRNA modification enzymes. These modifications are important for the functions of the tRNAs and for their correct folding. Many of the modifications are methylations of the bases or the ribose. Four families of tRNA methyltransferases are known, among which the SPOUT superfamily. Proteins of this superfamily are characterised by a C-terminal topological knot where the methyl donor is bound. With the exception of the monomeric Trm10, all known SPOUT proteins are dimeric and have an active site composed of residues of both protomers. Interestingly, depending on the organism, the same modification can be catalysed by completely unrelated enzymes. On the other hand, homologous enzymes can have different specificities or/and activities. These differences are well illustrated for the TrmJ and Trm10 enzymes.<p>In the first part of this work we have identified the TrmJ enzyme of Sulfolobus acidocaldarius (the model organism of hyperthermophilic Crenarchaeota) which 2’-O-methylates the nucleoside at position 32 of tRNAs. This protein belongs to the SPOUT superfamily and is homologous to TrmJ of the bacterium Escherichia coli. A comparative study shows that the two enzymes have different specificities for the nature of the nucleoside at position 32 as well as for their tRNA substrates. To try to understand these shifts of specificity at a molecular level we solved the crystal structure of the SPOUT domains of the two TrmJ proteins.<p>In the second part of this work, we have determined the crystal structure of the Trm10 protein of S. acidocaldarius. This is the first structure of a 1-methyladenosine (m1A) specific Trm10 and also the first structure of a full length Trm10 protein. The Trm10 protein of S. acidocaldarius is distantly related to its yeast homologues which are 1-methylguanosine (m1G) specific. To understand the difference of activity between the Trm10 enzymes, we compared the yeast and the S. acidocaldarius Trm10 structures. Remarkably several Trm10 proteins (such as Trm10 of Thermococcus kodakaraensis) are even able to form both m1A and m1G. To understand the capacity of the T. kodakaraensis protein to methylate A and G, a mutational study was initiated./Lors de la traduction, les ARN de transfert (ARNt) jouent le rôle crucial d’adaptateurs entre l’ARN messager et les acides aminés. Les ARNt sont transcrits sous forme de pré-ARNt qui doivent être maturés. Lors de cette maturation, plusieurs nucléosides sont modifiés. Un grand nombre de ces modifications sont des méthylations des bases ou du ribose. Quatre familles d’ARNt méthyltransferases sont actuellement connues, dont la superfamille des SPOUT. Les membres de cette superfamille sont caractérisés par un nœud dans la chaîne polypeptidique du côté C-terminal. C’est au niveau de ce nœud que se lie la S-adénosylméthionine qui est le donneur de groupement méthyle. A l’exception de Trm10 qui est monomérique, toutes les protéines SPOUT connues sont dimériques et leur site actif est formé de résidus provenant des deux protomères. Selon l’espèce, une même modification peut être formée à la même position dans la molécule d’ARNt par des enzymes qui appartiennent à des familles différentes. A l’opposé, des enzymes homologues peuvent présenter des spécificités ou des activités différentes.<p>Au cours de ce travail, nous avons identifié l’enzyme TrmJ de Sulfolobus acidocaldarius (l’organisme modèle des Crénarchées hyperthermophiles) qui méthyle le ribose du nucléoside en position 32 des ARNt. Cette protéine est un homologue de l’enzyme TrmJ de la bactérie Escherichia coli. L’étude comparative que nous avons réalisée a révélé que ces deux enzymes présentent une différence de spécificité pour la nature du nucléoside en position 32 ainsi que pour les ARNt substrats. Afin de comprendre ces différences de spécificité au niveau moléculaire, les structures des domaines SPOUT des deux TrmJ ont été déterminées et comparées.<p>En parallèle, nous avons résolu la structure cristalline de la protéine Trm10 de S. acidocaldarius. C’est la première structure disponible d’un enzyme Trm10 formant de la 1-méthyladénosine (m1A). C’est aussi la première structure complète d’une protéine Trm10. Les enzymes homologues des levures Saccharomyces cerevisiae et Schizosaccharomyces pombe qui n’ont que peu d’identité de séquence avec l’enzyme de S. acidocaldarius, forment de la 1-méthylguanosine (m1G). Dans le but de comprendre comment ces enzymes homologues peuvent présenter des activités différentes, leurs structures ont été comparées. De manière surprenante, certains homologues de Trm10 (comme l’enzyme de l’Euryarchée Thermococcus kodakaraensis) sont capables de former du m1A et du m1G. Afin de mieux comprendre comment ces protéines sont capables de méthyler deux types de bases, nous avons initié l’étude de l’enzyme Trm10 de T. kodakaraensis par mutagenèse dirigée.<p><p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

La maturation des ARNt en 5' est réalisée par RNase P. C'est un ribozyme chez les bactéries, les fungi et les nuclei des mammifères et un enzyme protéique dans les plantes ou des organelles des mammifères qui s’appelle PRORP. Il y a trois PRORP dans A. thaliana. PRORP contiennent deux domaines : un domaine PPR qui reconnaît spécifiquement des séquences d'ARN et un domaine nucléase qui assure la coupure endonucléolytique 5' des précurseurs d’ARNt. Pendant ma thèse j'ai pu montré par des méthodes biophysiques et structurales comme SRCD et SAXS que PRORP1 et 2 sont composées en majorité des hélices alpha Elles ont un rayon de giration de 33 Å et contiennent deux domaines distincts avec et une dimension maximale de 110 Å. Pour le complex entre un substrat d'ARNt et PRORP une constante de dissociation de 1 uM a pu être confirmé par la microcalorimétrie, la thermophorèse et l'ultracentrifugation analytique. Ces analyses nous ont permis de construire un modèle PRORP et un substrat d'ARNt. / RNase P cleaves 5’ leaders of precursor tRNAs. RNase P is a ribozyme in bacteria, fungi and animal nuclei and a protein in animal organelles, plants and many other organism. There are three PRORPs in A. thaliana. MALS, SRCD and SAXS provided first structural information: 1) PRORPs are monomers in solution. 2) PRORP 1-2 have a high alpha-helical content. 3) PRORPs are composed of two distinct domains with a radius of gyration of 33 A. These results together with homology modelling enabled us to build a first model of PRORPs in complex with tRNA. Using three different methods, isothermal titration calorimetry, microscale thermophoresis and analytical ultracentrifugation, a binding constant of about 1 µM could be determined for the system PRORP2mDD and L5T0 tRNA. This helped us conducting a SAXS experiment taking into account the low resolution affinity and designed to provide the direct structural data of a complex of proteinaceous RNase P with a substrate tRNA.

Des petits ARN non codants dérivant d'ARN de transfert (tRF) ont été identifiés dans tous les domaines de la vie, et de plus en plus de fonctions importantes leur sont attribuées chez de nombreux organismes. Dans ce travail mené sur la plante modèle Arabidopsis, nous avons d’abord montré que la population en tRF varie en fonction des tissus et des conditions de stress. Concernant leur biogenèse, les endoribonucléases responsables du clivage des ARNt ont été identifiées, il s'agit des RNases T2, RNS1, 2 et 3. Afin de réaliser une étude structure/fonction, une approche d’expression en système de levure a été initiée pour permettre l’obtention de quantité suffisante de RNS1 purifiée. L’étude des fonctions des tRF montre que certains d’entre eux sont associés à AGO1, qu'ils semblent cibler entre-autres des éléments transposables et qu’ils pourraient avoir une localisation nucléaire. Enfin, deux tRF, le tRF-5D (Ala) et le tRF-5D (Asn) inhibent efficacement la traduction in vitro. Une association de tRF-5D (Ala) aux polyribosomes de plantules d'Arabidopsis a pu être visualisée, suggérant que certains tRF puissent agir en tant que régulateur global de la traduction. / Small non-coding RNAs derived from transfer RNAs (tRFs) have been identified in all domains of life, and more and more important functions are attributed to them in many organisms. In this work on the model plant Arabidopsis, we first showed that the tRF population varies according to tissues and stress conditions. With regard to their biogenesis, the endoribonucleases responsible for tRNA cleavage were identified, it is the RNases T2, RNS1, 2 and 3. In order to carry out a structure / function analysis, heterologous expression in yeast has been developed with the hope to get sufficient amount of purified RNS1. The question of tRF functions has also been studied. It has been shown that some tRFs are associated with AGO1, that they often seem to target transposable elements and could have a nuclear localization. Finally, the study of the involvement of the tRFs in the regulation of translation was tackled. Two tRFs, tRF-5D (Ala) and tRF-5D (Asn) efficiently inhibit translation in vitro. An association of tRF-5D (Ala) with polyribosomes of Arabidopsis seedlings could be visualized suggesting that some plant tRFs could as global regulator of the translation process.

Le travail effectué dans cette thèse présente une nouvelle approche de la théorie du code circulaire dans les gènes qui a été initiée en 1996. Cette approche consiste à analyser les motifs construits à partir de ce code circulaire, ces motifs particuliers sont appelés motifs de code circulaire. Ainsi, nous avons développé des algorithmes de recherche pour localiser les motifs de code circulaire dans les séquences d'acides nucléiques afin de leur trouver une signification bioinformatique. En effet, le code circulaire X identifie dans les gènes est un ensemble de trinucleotides qui a la propriété de retrouver, synchroniser et maintenir la phase de lecture. Nous avons commencé notre analyse avec le centre de décodage du ribosome (ARNr) qui est une région majeure dans le processus de traduction des gènes aux protéines. Puis, nous avons étendu les résultats obtenus avec le ribosome aux ARN de transfert (ARNt) pour étudier les interactions ARNr-ARNt. Enfin, nous avons généralisé la recherche de motifs de code circulaire X dans l'ADN aux chromosomes d'eucaryotes complets. / The work done in this thesis presents a new direction for circular code identified in 1996 by analysing the motifs constructed from circular code. These particular motifs are called circular code motifs. We applied search algorithms to locate circular code motifs in nucleic acid sequences in order to find biological significance. In fact, the circular code X, which was found in gene sequences, is a set of trinucleotides that have the property of reading frame retrieval, synchronization and maintenance. We started our study in the ribosomal decoding centre (rRNA), an important region involved in the process of translating genes into proteins. Afterwards, we expanded our scope to study the interaction of rRNA through the X circular code. Finally, we search for the X circular code motifs in the complete DNA sequences of chromosomes of the eukaryotic genomes. This study introduced new properties to the circular code theory.

No description available.