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tRNAs spielen eine zentrale Rolle in jedem bekannten Organismus. Sie agieren als Adaptoren, um den Code der Nukleinsäuren in eine funktionale Aminosäuresequenz zu übersetzen. Ihre Struktur folgt einer hoch konservierten Kleeblattform, die aus dem D-Arm, dem Anticodonarm und –loop, dem TΨC-Arm und dem Akzeptorstamm gebildet wird. 5ʹ-und 3ʹ-Ende bilden den Akzeptorstamm, wobei das 3ʹ-Ende mit dem Diskriminator und dem universalen CCA-Triplett um vier Nukleotide herausragt. Das CCA-addierende Enzym führt die Addition dieses CCA-Tripletts durch und ist ubiquitär in Bakterien, Archaeen und Eukaryoten vorhanden. Für diese Arbeit wurden zwei ungewöhnliche Mitglieder der CCA-addierenden Enzyme untersucht: das CCA-addierende Enzym aus dem Nematoden Romanomermis culicivorax als auch das bifunktionale CCA-addierende Enzym aus dem Bakterium Escherichia coli. In den Mitochondrien des Nematoden Romanomermis culicivorax sind tRNAs codiert, die von der Kleeblattstruktur abweichen. Es wurden die Anpassungen des CCA-addierenden Enzyms an diesen abweichenden tRNA Pool charakterisiert. Der zweite Teil dieser Arbeit betrachtet eine Phosphodiesterase-Domäne, die HD Domäne, in den CCA-addierenden Enzymen der Gammaproteobakterien. In vitro zeigt diese HD Domäne die Abspaltung eines 2ʹ,3ʹ-cyclischen Phosphates vom 3ʹ-Ende von tRNA Strukturen. Weiterhin wurde in in vivo Experimenten die Funktion der HD Domäne des CCA-addierenden Enzyms aus Escherichia coli betrachtet. Dabei wurde beobachtet, dass in Escherichia coli der RNA-Pool mit 2ʹ,3ʹ-cyclischen Phosphaten dynamisch auf Nährstoffmangel reagiert. Insbesondere der tRNA-Pool und deren Fragmente zeigten interessante Veränderungen, wenn Escherichia coli hohe Zelldichten erreichte oder von einem nährstoffreichen Medium auf Minimalmedium versetzt wurde.

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The mitochondrial genome of the nematode Romanomermis culicivorax encodes for miniaturized hairpin-like tRNA molecules that lack D- as well as T-arms, strongly deviating from the consensus cloverleaf. The single tRNA nucleotidyltransferase of this organism is fully active on armless tRNAs, while the human counterpart is not able to add a complete CCA-end. Transplanting single regions of the Romanomermis enzyme into the human counterpart, we identified a beta-turn element of the catalytic core that—when inserted into the human enzyme—confers full CCA-adding activity on armless tRNAs. This region, originally identified to position the 30 -end of the tRNA primer in the catalytic core, dramatically increases the enzyme’s substrate affinity. While conventional tRNA substrates bind to the enzyme by interactions with the T-arm, this is not possible in the case of armless tRNAs, and the strong contribution of the beta-turn compensates for an otherwise too weak interaction required for the addition of a complete CCA-terminus. This compensation demonstrates the remarkable evolutionary plasticity of the catalytic core elements of this enzyme to adapt to unconventional tRNA substrates.

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Le rôle des deux paires de bases universelles inverse Hoogsteen U : A ( RHUAs ) présentent chez les ARNt standards , une dans la boucle T et l'autre dans le noyau de la forme en L , a été étudiée. Pour chacun des RHUAs , un criblage génétique spécialisé in vivo chez les bactéries , le système suppresseur ambre ( pour l'étude de la RHUA dans la boucle T ) et le système d'ARNt de la sélénocystéine ( tRNASec ) ( pour l'étude de la RHUA dans le noyau ) , ont été utilisé pour générer des variants fonctionnels à partir de multiples librairies combinatoires . Ces variants ont ensuite été séquencé et soumis à une analyse systématique qui comprend la modélisation informatique et un type d'analyse phylogénétique. Les résultats du système suppresseur ambre ont montré un ensemble de variants fonctionnels qui ne nécessitent pas le motif RHUA dans la boucle T et qui ont remplacé la méthode standard de l'interaction entre les boucles D et T avec une double hélice interboucle , ILDH . D'autres études ont abouti à la détermination d'un modèle In silico de l'alternative à la norme standard de la boucle T, sous le nom de type III . Les résultats du système tRNASec ont révélé que pour cette ARNt exceptionnel, l'absence de RHUA ( dans le noyau ) assure une flexibilité accrue qui est spécifiquement nécessaire pour la fonction de tRNASec . Ainsi, les ARNt standards , à la différence de tRNASec , avec la présence universelle de RHUA dans le noyau , a été naturellement sélectionnée pour être rigide . Pris ensemble, la RHUA joue un rôle essentiel dans la stabilisation des interactions tertiaires. / The role of two universally present reverse Hoogsteen U:A base pairs (RHUAs) in the T-loop and in the core of the L-shape of standard tRNA was studied. To study each of the RHUAs, bacterial in vivo genetic screens were used including the amber suppressor system (for the study of the RHUA in the T-loop) and the selenocysteine tRNA(tRNASec) system (for the study of the RHUA in the core). These screens generated functional variants from multiple combinatorial libraries. These variants were subsequently sequenced and subjected to a systematic analysis which included computer modeling and a type of phylogenetic analysis. The results from the amber suppressor system showed a set of functional variants which did not require the RHUA motif in the T-loop, and had replaced the standard way of interaction between the D and T loops with an interloop double helix, ILDH. Further study culminated in the determination of an insilico model of the alternative to the standard T-loop known as type III. The results from the tRNASec system revealed that for this exceptional tRNA, the absence of RHUA (in the core) ensures an enhanced flexibility that is specifically required for tRNASec function. Thus standard tRNAs, unlike tRNASec, with the universal presence of RHUA in the core have been naturally selected to be rigid. Taken together, RHUA plays an essential role in the stabilization of tertiary interactions.

Ground state destabilization is a hypothesis to explain enzyme catalysis. The most popular interpretation of it is the entropic effect, which states that enzymes accelerate biochemical reactions by bringing the reactants to a favorable position and orientation and the entropy cost of this is compensated by enthalpy of binding. Once the enzyme-substrate complex is formed, the reaction could proceed with negligible entropy cost. Deamination of cytidine catalyzed by E.coli cytidine deaminase appears to agree with this hypothesis. In this reaction, the chemical transformation occurs with a negligible entropy cost and the initial binding occurs with a large entropy penalty that is comparable to the entropic cost of the uncatalyzed reaction. Our calculations revealed that this reaction occurs with different mechanisms in the cytidine deaminase and water. The uncatalyzed reaction involves a concerted mechanism and the entropy cost of this reaction appears to be dominated by the reacting fragments and first solvation shell. The catalyzed reaction occurs via a stepwise mechanism in which a hydroxide ion acts as the nucleophile. In the active site, the entropy cost of hydroxide ion formation is eliminated due to pre-organization of the active site. Hence, the entropic effect in this reaction is due to a pre-organized active site rather than ground state destabilization. In the second part of this thesis, we investigated peptide bond formation and peptidyl-tRNA hydrolysis at the peptidyl transferase center of the ribosome. Peptidyl-tRNA hydrolysis occurs by nucleophilic attack of a water molecule on the ester carbon of peptidyl-tRNA. Our calculations showed that this reaction proceeds via a base catalyzed mechanism where the A76 O2’ is the general base and activates the nucleophilic water. Peptide bond formation occurs by nucleophilic attack of the α-amino group of aminoacyl-tRNA on the ester carbon of peptidyl-tRNA. For this reaction we investigated two mechanisms: i) the previously proposed proton shuttle mechanism which involves a zwitterionic tetrahedral intermediate, and ii) a general base mechanism that proceeds via a negatively charged tetrahedral intermediate. Although both mechanisms resulted in reasonable activation energies, only the proton shuttle mechanism found to be consistent with the pH dependence of peptide bond formation.