The problem of forming a glycosidic bond between ribose and the free nucleoside bases to produce beta-nucleosides under plausible prebiotic conditions is commonly referred to in origin of life research as The Nucleoside Problem. The lack of a general solution to this problem currently represents one of the largest stumbling blocks to the RNA world hypothesis and many other theories regarding the origin of life. Over thirty years ago the purine nucleosides were successfully synthesized by drying the fully-formed bases and ribose together in the presence of divalent metal ion salts. However, glycosidic bond formation by the pyrimidine bases has never been achieved under similar reaction conditions. This thesis describes the first plausible prebiotic synthesis of a pyrimidine nucleoside, demonstrated with the pyrimidine base analogue 2-pyrimidinone. Information provided by nucleoside-formation reaction involving 2-pyrimidinone and related pyrimidine bases should provide valuable insights into the possible mechanism by which glycosidic bond formation was accomplished on the prebiotic Earth.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Pseudouridine is the most common RNA modification found in all forms of life. The exact role pseudouridines play in the cell is still relatively unknown. However, its extensive incorporation in functionally important areas of the ribosome and the fitness advantage provided to cells by pseudouridines implies that its presence is important for the cell. The enzymes responsible for this modification, pseudouridine synthases, vary greatly in substrate recognition mechanisms, but all enzymes supposedly share a universally conserved catalytic mechanism. Here, I analyze the kinetic mechanisms of pseudouridylation utilized by the exemplary pseudouridine synthase RluA in order to compare it with the previously determined rate of pseudouridylation of the pseudouridine synthase TruB. My results demonstrate that RluA has the same uniformly slow catalytic step as previously determined for TruB and TruA. This constitutes the first step towards identifying the catalytic mechanism of the pseudouridine synthase family. Additionally, it was my aim to identify the major determinants for RNA binding by pseudouridine synthases. By measuring the dissociation constants (KD) for substrate and product tRNA by nitrocellulose filtration assays, I showed that both tRNA species could bind with similar affinities. These binding studies also revealed that TruB’s interaction with the isolated T-arm is the major contact site contributing to the affinity of the enzyme to RNA. Finally, a new contact between tRNA and TruB’s PUA domain was identified which was not observed in the crystal structure. In summary, my results provide new insight into the common catalytic step of pseudouridine synthases and the specific interactions contributing to substrate binding by the enzyme TruB. These results will enable future studies on the kinetic mechanism of pseudouridine synthases, in particular the kinetics of substrate and product binding and release, as well as on the chemical mechanism of pseudouridine formation. / xi, 122 leaves : ill. (some col.) ; 29 cm

Diversity-oriented synthesis (DOS) involves the synthesis of several synthetic targets by transforming a collection of structurally simple and similar starting materials into a collection of structurally more complex and diverse products. This thesis describes the elaboration of a 6,6-spiroacetal scaffold to incorporate biologically useful moieties, in particular nucleobases, triazoles and amino acids, thus generating a collection of novel hybrid structures. The research reported, herein, focused on the synthesis of spiroacetal-nucleosides, triazoles and amino acids bearing a C8′-hydroxymethyl substituent.

Diversity-oriented synthesis (DOS) involves the synthesis of several synthetic targets by transforming a collection of structurally simple and similar starting materials into a collection of structurally more complex and diverse products. This thesis describes the elaboration of a 6,6-spiroacetal scaffold to incorporate biologically useful moieties, in particular nucleobases, triazoles and amino acids, thus generating a collection of novel hybrid structures. The research reported, herein, focused on the synthesis of spiroacetal-nucleosides, triazoles and amino acids bearing a C8′-hydroxymethyl substituent.

Diversity-oriented synthesis (DOS) involves the synthesis of several synthetic targets by transforming a collection of structurally simple and similar starting materials into a collection of structurally more complex and diverse products. This thesis describes the elaboration of a 6,6-spiroacetal scaffold to incorporate biologically useful moieties, in particular nucleobases, triazoles and amino acids, thus generating a collection of novel hybrid structures. The research reported, herein, focused on the synthesis of spiroacetal-nucleosides, triazoles and amino acids bearing a C8′-hydroxymethyl substituent.

Diversity-oriented synthesis (DOS) involves the synthesis of several synthetic targets by transforming a collection of structurally simple and similar starting materials into a collection of structurally more complex and diverse products. This thesis describes the elaboration of a 6,6-spiroacetal scaffold to incorporate biologically useful moieties, in particular nucleobases, triazoles and amino acids, thus generating a collection of novel hybrid structures. The research reported, herein, focused on the synthesis of spiroacetal-nucleosides, triazoles and amino acids bearing a C8′-hydroxymethyl substituent.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [251]-261).