In this project a number of peptidyl transferase antibiotics were studied, specifically a group of aminohexose cytosine nucleoside antibiotics and their interaction with a selected number of highly conserved ribonucleic acid (RNA) motifs, designed to represent their possible binding site within the ribosome. This group of antibiotics shows a wide range of interesting properties, including antiviral and anti-tumour activity, and as they bind to a particularly conserved region in the ribosome, they are likely to be difficult for microorganisms to develop resistance to. It is hoped that once the mechanism of action of these antibiotics is better understood, that modifications to the antibiotics can be effectively made to create new or hybrid antibiotics with more selective antibacterial, or indeed antiviral or anti-tumour properties. The nuclear magnetic resonance (NMR) structure of the RNA binding, peptidyl tranferase inhibitor antibiotics amicetin, blasticidin S and gougerotin, in their native solution states, have been successfully determined. The structures all exhibit a stable conformation, stabilised by intramolecular hydrogen bonds. Amicetin was observed to be folded, distinctly different from the linear, extended conformation of amicetin previously determined by X-ray crystallography. The structure of blasticidin S was found to be very similar to its X-ray crystal structure. Gougerotin was shown to form a similar conformation to blasticidin S, save that the end chain of gougerotin was bent at right angles to the rest of the molecule, forming a structure similar to that of the major bound X-ray crystal structure of blasticidin S. All the solution structures showed a similar conformation in the analogous regions of their chemical structure, suggesting that hybrid antibiotics could be produced.Two highly conserved RNA motifs of Halobacterium halobium (H. h.) and Escherichia coli (E. coli) 23S ribosomal RNAs were chosen to investigate their interaction with amicetin. The NMR structure of the H. h. and E. coli. 29-mer RNA motifs have been determined; the motifs both form well folded A-form RNA conformations. The E. coli NMR structure differs from the X-ray crystal structure of the motif contained within the ribosome, as a highly conserved adenine residue, which resides in a bulge strongly implicated with amicetin binding, folds into the helix as opposed to being flipped out. Instead, an adjacent cytosine residue partially flips out; whereas in the crystal structure, it is folded within the helix. The NMR stuctures of the H. h. motif differs from the X-ray crystal structure of the motif, contained within the ribosome, as none of the bases are flipped out and a number of non-canonical base pairs are formed in the solution structure. To continue this study, a fully 13C and 15N isotopically labelled version of the H. h. RNA sample has been partially assigned, and an initial structure determination has been performed, using ultra high field 1 GHz spectroscopy.Addition of amicetin to both the H. h. and E. coli 29-mer RNA samples were accompanied by discrete changes to the spectra, suggesting weak interaction between the two components. These can be qualitatively interpreted to changes induced in the local conformation of the RNA motifs and the amicetin arising from the formation of a complex, between the amicetin and the bulge region of the particular motif.

Les interactions entre les squelettes sucre-phosphate de nucléotides jouent un rôle important dans la stabilisation des structures tertiaires de larges molécules d’ARN. Elles sont régies par des règles particulières qui gouverne leur formation mais qui jusque là demeure quasiment inconnues. Un élément structural d’ARN pour lequel les interactions sucre-phosphate sont importantes est le motif d’empaquetage de deux doubles hélices d’ARN le long du sillon mineur. Ce motif se trouve à divers endroits dans la structure du ribosome. Il consiste en deux doubles hélices interagissant de manière à ce que le squelette sucre-phosphate de l’une se niche dans le sillon mineur de l’autre et vice versa. La surface de contact entre les deux hélices est majoritairement formée par les riboses et implique au total douze nucléotides. La présente thèse a pour but d’analyser la structure interne de ce motif et sa dépendance de stabilité résultant de l’association optimale ou non des hélices, selon leurs séquences nucléotidiques. Il est démontré dans cette thèse qu’un positionnement approprié des riboses leur permet de former des contacts inter-hélices, par l’entremise d’un choix particulier de l’identité des pairs de bases impliquées. Pour différentes pairs de bases participant à ce contact inter-hélices, l’identité optimale peut être du type Watson-Crick, GC/CG, or certaines pairs de bases non Watson-Crick. Le choix adéquat de paires de bases fournit une interaction inter-hélice stable. Dans quelques cas du motif, l’identité de certaines paires de bases ne correspond pas à la structure la plus stable, ce qui pourrait refléter le fait que ces motifs devraient avoir une liberté de formation et de déformation lors du fonctionnement du ribosome. / Although backbone-backbone interactions play an important role in stabilization of the tertiary structure of large RNA molecules, the particular rules that govern the formation of these interactions remain basically unknown. One RNA structural element for which the backbone-backbone interactions are essential is the along-groove packing motif. This motif is found in numerous locations in the ribosome structure; it consists of two double helices arranged such that the backbone of one helix is packed in the minor groove of the other helix and vice versa. The contact area between the two helices is mostly formed by riboses and totally involves twelve nucleotides. Here we analyze the internal structure of the along-groove packing motif and the dependence of stability of the association of the helices on their nucleotide sequences. We show that the proper positioning of the riboses that allows them to form inter-helix contacts is achieved through the particular choice of the identities of the base pairs involved. For different base pairs participating in the inter-helix contacts the optimal identities can be Watson-Crick, GC/CG, or certain non-Watson-Crick base pairs. The proper choice of the base pairs provides for the stable inter-helix interaction. In some cases of the motif, the identities of certain base pairs do not correspond to the most stable structure, which may reflect the fact that these motifs should break and form during the ribosome function.