The presence and synthesis of transfer RNA (tRNA) is highly conserved in all organisms and a lot of genetic material is dedicated to its synthesis. tRNA contains a large number of modified nucleosides and several diverse functions have been found but much about their function is still unknown. By using a novel frameshifting system to select for tRNA modification mutants, new mutations were isolated and subsequently analyzed. This thesis examines the synthesis and function of a subset of tRNA modifications that have a sulfur (thio) -group as part of the modification. The isc operon encodes for proteins synthesizing iron sulfur centers ([Fe-S]) that are a part of the active site of many key enzymes in the cell and the thiolated nucleosides are dependant on a functional iron sulfur gene (iscS) for their synthesis. By studying thiolated tRNA it is not only possible to learn more about the synthesis of the modifications themselves, but also about the synthesis of [Fe-S] clusters. Based on an analysis of mutations in three of the isc operon genes (iscS, iscU, and iscA), a two-model pathway is proposed for the synthesis of Salmonella enterica Serovar Typhimurium thiolated tRNA modifications. The interactions of IscS with other proteins in the tRNA modification thiolation pathways suggest a more complex sulfur relay than had previously been envisioned. Some of the specificities and the effect of an iscA mutant on the levels of tRNA modifications lead to an examination of the role of IscA in [Fe-S] formation and its importance for tRNA modifications.

No description available.

<p>This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (p<i>K</i>_a) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic <i>NE</i>-type (P = 37° ± 27°, Φ_m = 25° ± 18°) for 1′,2′-constrained nucleosides, and <i>N</i>-type (P = 19° ± 8°, Φ_m = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)₂, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).</p>

This thesis is based on six research publications concerned with (i) study of the molecular structures and dynamics of modified nucleosides; (ii) investigation of the effect of incorporation of modified nucleosides on the structure of DNA; (iii) examination of the effect of the sugar modifications on the pseudo-aromatic properties (pKa) of the nucleobases; (iv) analysis of the effect of the CH-π interactions on the relative stability of the DNA-RNA hybrid duplexes. The structural stability of the nucleic acids as well as their behavior in molecular recognition is dominated by hydrogen bonding and stacking interactions beside other non-covalent interactions. Naturally occurring nucleosides are found to have some specific functions. Modifications of nucleic acids, followed by studies of the resulting structural, chemical and functional changes, contribute to an understanding of their role in various biochemical processes, such as catalysis or gene silencing. In papers I-III, analysis of the structures of modified thymidine nucleosides with 1′,2′-(oxetane or azetidine) and 2′,4′-(LNA, 2′-amino LNA, ENA, and Aza-ENA) conformationally constrained sugar moieties, and dynamics of the modified nucleosides by NMR, ab initio, and molecular dynamics simulations are discussed. Based on whether the modification leads to 1′,2′- or 2′,4′- constrained sugar moieties, it is found that they fall into two distinct categories characterized by their respective internal dynamics of the glycosidic and backbone torsions as well as by their characteristic NE-type (P = 37° ± 27°, Φm = 25° ± 18°) for 1′,2′-constrained nucleosides, and N-type (P = 19° ± 8°, Φm = 48° ± 4°) for 2′,4′-constrained systems, respectively. Moreover, each group has different conformational hyperspace accessible. The effect of the incorporation of 1′,2′-oxetane locked thymidine nucleoside on the structure and dynamics of the Dickerson-Drew dodecamer, d(CGCGAATTCGCG)2, determined by NMR, is discussed in the paper IV. It shows that the incorporation of oxetane locked T into the dodecamer has made local structural deformations and perturbation in base pairing, where the modification is included. The modulations of physico-chemical properties of the nucleobases in nucleotides by the C2′-modification of the sugar (paper V), 5′-phosphate group, and the effect of constrained pentofuranosyl moiety (sugar, paper III) have been studied. CH-π interactions between the methyl group of thymidine and the neighboring aromatic nucleobase are shown to increase the relative stability of the DNA-RNA hybrid duplexes over the isosequential RNA-DNA duplexes or vice versa (paper VI).

Transfer RNAs (tRNA) function as adaptor molecules in the translation of mRNA into protein. These adaptor molecules require modifications of a subset of their nucleosides for optimal function. The most frequently modified nucleoside in tRNA is position 34 (wobble position), and especially uridines present at this position. Modified nucleosides at the wobble position are important in the decoding process of mRNA, i.e., restriction or improvement of codon-anticodon interactions. This thesis addresses the functional aspects of the wobble uridine modifications. The Saccharomyces cerevisiae Elongator complex consisting of the six Elp1-Elp6 proteins has been proposed to participate in three distinct cellular processes; elongation of RNA polymerase II transcription, regulation of polarized exocytosis, and formation of modified wobble nucleosides in tRNA. In Paper I, we show that the phenotypes of Elongator deficient cells linking the complex to transcription and exocytosis are counteracted by increased level of and . These tRNAs requires the Elongator complex for formation of the 5-methoxycarbonylmethyl (mcmlnGUUGsmcm25tRNALysUUUsmcm25tRNA5) group of their modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U). Our results therefore indicate that the relevant function of the Elongator complex is in formation of modified nucleosides in tRNAs and the defects observed in exocytosis and transcription are indirectly caused by inefficient translation of mRNAs encoding gene products important for these processes. The lack of defined mutants in eukaryotes has led to limited understanding about the role of the wobble uridine modifications in this domain of life. In Paper II, we utilized recently characterized mutants lacking the 2-thio (s2) or 5-carbamoylmethyl (ncm5) and mcm5 groups to address the in vivo function of eukaryotic wobble uridine modifications. We show that ncm5 and mcm5 side-chains promote reading of G-ending codons, and that presence of a mcm5 and an s2 group cooperatively improves reading of both A- and G-ending codons. Previous studies revealed that a S. cerevisiae strain deleted for any of the six Elongator subunit genes shows resistance towards a toxin (zymocin) secreted by the dairy yeast Kluyveromyces lactis. In Paper III, we show that the cytotoxic γ subunit of zymocin is a tRNA endonuclease that target the anticodon of mcm5s2U34 containing tRNAs and that the wobble mcm5 modification is required for efficient cleavage. This explains the γ-toxin resistant phenotype of Elongator mutants which are defective in the synthesis of the mcm5 group.

Transfer RNA (tRNA) is the adaptor molecule responsible for bringing the correct amino acid to the ribosome during protein synthesis. tRNA contains a number of modified nucleosides, which are derivatives of the four normal nucleosides. A great variety of modifications are found in the anticodon loop, especially at the first (wobble) position of the anticodon. According to Crick’s wobble hypothesis, a uridine at the wobble position of tRNA recognize codons ending with A and G. Uridine-5-oxyacetic acid (cmo5U34), found at the wobble position of six species of tRNA in Salmonella enterica, have been predicted to expand the codon recognition of uridine to include U-ending, but not C-ending codons. To study the function of cmo5U34 we have identified two genes, cmoA and cmoB, which are required for the synthesis of cmo5U34 in tRNA. We have shown that the proline, alanine and valine tRNAs containing cmo5U34 are capable of reading codons ending with any of the four nucleotides, while the threonine tRNA is not, and the importance of having cmo5U is different for the different tRNAs. In addition, we found that cmo5U is important for efficient reading of G-ending codons, which is surprising considering the wobble hypothesis, which states that uridine should read G-ending codons. The dominant +1 frameshift suppressor sufY suppresses the hisC3737 +1 frameshift mutation. We have demonstrated that sufY induces frameshifting at CCC-CAA (Pro-Gln), when tRNAPro[cmo5UGG] occupies the P-site. sufY mutants accumulate novel modified nucleosides at the wobble position of tRNAs that should normally have (c)mnm5s2U34. The presence of an extra sidechain (C10H17) on the wobble nucleoside of tRNAGln[(c)mnm5s2U] leads to slow decoding of CAA codons, inducing a translational pause that allows the P-site peptidyl-tRNAPro[cmo5UGG] to slip into the +1 frame. We have characterized 108 independent frameshift suppressor mutants in the gene encoding tRNAPro[cmo5UGG]. The altered tRNAs are still able to read all four proline codons in the A-site, but induce frameshifts after translocation into the P-site. Some of the mutations are in regions of the tRNA that are involved in interactions with components of the P-site. We hypothesize that the ribosomal P-site keeps a “grip” of the peptidyl-tRNA to prevent loss of the reading frame.

No description available.

Development of chiral ligands capable of achieving high selectivity for various asymmetric catalytic reactions has been an important aim of both academia and industry. Nature is capable to selectively catalyze chemical reactions by using enzymes. An ideal catalyst would combine the selectivity of nature and the reactivity of man-made catalysts based on transition metal complexes. The two biomolecules chosen to achieve this are DNA and PNA. DNA is a chiral molecule with high binding selectivity towards small molecules and has been used as ligand for asymmetric catalysis. PNA is an achiral structural analogue of DNA that can form duplexes with DNA. To produce DNA based catalysts it is necessary to introduce a ligand such as a phosphine that will strongly coordinate to transition metals. To achieve this, functionalized linkers need to be introduced into a DNA strand, to covalently couple the phosphine moiety at a specific location of the DNA strand. Amine linkers and several modified nucleosides have been prepared containing thiol and amine functionalities and some of them were successfully introduced into DNA strands to function as linkers for the introduction of phosphine functionalities. Those strands were purified and an adequate procedure was developed for their analysis by MALDI-TOF. Diphenylphosphino carboxylic acids have been coupled to amine modified deoxyuridines by amide bond formation. The same coupling method has been used for oligonucleotides. DNA strands containing phosphine moieties were characterized by MALDI-TOF and ³¹P NMR spectrometry. ³¹P NMR spectroscopy was also used to confirm coordination of a phosphine modified 15-mer to [PdCl(η³-allyl)]₂. The phosphine modified nucleobases were also tested as ligands for palladium catalyzed allylic alkylation and allylic amination with diphenylallyl acetate as substrate although no enantioselectivity was observed. A PNA monomer was also modified with a bidentate sulfur protected phosphine and successfully introduced into a short PNA strand using manual solid phase synthesis. This strand was analyzed by MALDI-TOF. Moreover, preliminary studies were performed to test the use of aptamers as scaffolds for targets containing a ligand functionality.

Cela fait plus de quarante ans que la plupart des modifications des ARNt ont été découvertes mais ce n’est que récemment que les gènes correspondants ont pu être identifié. La modification N6-threonylcarbamoyl adénosine (t6A) est universelle et se trouve à la position 37, adjacente de l’anticodon, dans de nombreux ARNt. Les quatre gènes responsables de la synthèse de cette modification chez les bactéries furent découverts par des approches de génomique comparative mais uniquement deux de ces gènes sont universels, TsaC/Sua5 et TsaD/Kae1/Qri7. Des travaux récents ont révélé qu'il existait différentes voies enzymatiques pour la synthèse de cette modification selon domaine de la vie, les organelles et les espèces considérés. L'étude de ces variations est toujours en cours de caractérisation.Ce travail a identifié quatre autres protéines requises pour la synthèse de t6A dans les ARNt cytoplasmiques de levure (Bud32, Pcc1, Cgi121 et Gon7) et établi que seuls Sua5 et Qri7 sont requis pour modifier les ARNt mitochondriaux. La même enzyme, Sua5, effectue la première étape de la synthèse de t6A à la fois dans le cytoplasme et les mitochondries. Cette protéine peut être localisée dans les deux compartiments grâce à l’utilisation de sites d’initiation de la traduction différents. Cette étude a montré qu’une machinerie de synthèse minimale est requise pour la synthèse de t6A dans les mitochondries, potentiellement similaire à la machinerie présente dans le dernier ancêtre commun. Les rôles de cette modification complexe in vivo semblent également varier. Par exemple, t6A est indispensable chez les procaryotes, mais pas dans la levure. Les causes des phénotypes pléïotropes observés lors de la diminution ou l'absence de t6A ne sont pas encore entièrement comprises. Nous avons pu élucider certains des rôles joués par la modification t6A, en effectuant une analyse globale des erreurs de traduction observées en absence de cette modification par analyse des profils ribosomaux. Par exemple, il semble que la présence de t6A permet aux ARNt rares de concurrencer plus efficacement les ARNt abondants. La complexité et la diversité des voies de synthèse combiné à l’importance fonctionnelle et évolutive de cette modification ont fait de t6A une “décoration” des ARNt particulièrement fascinante à étudier. / The modification of tRNA has a rich literature of biochemical analysis going back more than 40 years; however, the genes responsible for the modifications have only been recently identified. Comparative genomic analysis has allowed for the identification of the genes in bacteria, and subsequent characterization of the enzymes, responsible for the modification N6-threonylcarbamoyladenosine (t6A) located at position 37, adjacent to the anticodon of tRNAs. While the modification is present in all domains of life, only two of the four enzymes responsible for biosynthesis machinery are conserved. In Eukaryotes, both cytoplasmic and mitochondrial tRNAs are modified with t6A, and previously only the two universally conserved members of the cytoplasmic t6A synthesis pathway, TsaC/Sua5 and TsaD/KaeI/Qri7 were known. Recent progress on deciphering the t6A synthesis pathways has revealed that different solutions have been adopted in different kingdoms, species, and organelles, and these variant pathways are still being characterized.This investigation identified the other four proteins required for cytoplasmic synthesis (Bud32, Pcc1, Cgi121, Gon7), and determined that only Sua5 and Qri7 are required for mitochondrial synthesis of t6A in yeast. The same enzyme, Sua5, performs the first step of t6A synthesis in both the cytoplasm and the mitochondria. It is targeted to both the cytoplasm and the mitochondria through the use of alternative, in-frame AUG translational start sites. This study showed that a minimum synthesis machinery is responsible for mitochondrial t6A, implicating a core set of enzymes from the LUCA.The roles of this complex modification in vivo also seem to vary. For example, t6A is essential in prokaryotes, but not in yeast. The causes of the observed pleiotropic phenotypes triggered by the reduction or absence of t6A synthesis enzymes are not yet fully understood. This work used ribosome profiling to map all translation errors occurring when t6A was absent. By examining ribosomal occupancy of every codon, this work indicates that t6A is helping rare tRNAs compete with high copy tRNAs. The complexity and diversity of the t6A pathway combined with the functional and evolutionary importance of this modification have made t6A a particularly fascinating “decoration” of tRNA to study.

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