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Structural and biophysical studies of RNase P and RNA Aptamers

WERNER, Andreas (2005) Structural and biophysical studies of RNase P and RNA Aptamers. Thèses de doctorat, Université Louis Pasteur.

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Résumé

The research in this Ph.D. thesis is part of an ongoing effort to improve our understanding of RNA structure and stability: In the first part, an RNA aptamer is studied in complex with its target, the Sec7 domain of intracellular protein cytohesin-1. Aptamers are in vitro selected RNA molecules that have an affinity rivaling that of monoclonal antibodies, possess interesting pharmacological properties and are a powerful tool for functional analysis in vivo. We also present a systematic structural investigation of how aptamers and their natural counterparts bind their ligands in order to delineate common principles. In the second part, we study the counterion-induced collapse of prokaryotic RNase P RNAs by UV melting, with the goal of optimizing secondary and tertiary structure for crystallization. We demonstrate the utility of this method in two test cases: First, thermodynamic parameters are extracted from the kissing complex, a typical tertiary interaction found in many RNAs. This leads to the discovery that the ionic strength dependence may give clues about the structural transitions observed. Second, we study the effect of mutations on a more complex RNA, the IRES site of hepatitis C virus, and correlate UV melting data to a dynamic equilibrium between alternative structures. The lessons learned from these experiments are then applied to RNase P to disrupt specific tertiary interactions to facilitate crystallization. The effect of these mutations is investigated using both UV melting and a novel, fluorescence-based approach. Finally, crystallization screens are constructed using methods of experimental design, and a new approach for crystallization in gels is developed.

Type d'EPrint:Thèse de doctorat
Informations complémentaires:Utilisateurs enregistrés uniquement. Pour tout renseignement, s'adresser au Service des thèses du SCD de Strasbourg
Sujets:CL Classification > DDC Dewey Decimal Classification > 500 Sciences de la nature et mathématiques > 570 Sciences de la vie. Biologie. Biochimie > 572 Biochimie > 572.8 Génétique biochimique
Classification Thèses Unistra > Santé > Sciences de la vie, biologie, biochimie > 570 Sciences de la vie. Biologie. Biochimie > 572 Biochimie > 572.8 Génétique biochimique

CL Classification > DDC Dewey Decimal Classification > 500 Sciences de la nature et mathématiques > 570 Sciences de la vie. Biologie. Biochimie > 572 Biochimie > 572.7 Enzymes
Classification Thèses Unistra > Santé > Sciences de la vie, biologie, biochimie > 570 Sciences de la vie. Biologie. Biochimie > 572 Biochimie > 572.7 Enzymes

UNERA Classification UNERA > DISC Discipline UNERA > DISC-16 Sciences de la vie et de la santé, psychologie
UNERA Classification UNERA > ACT Domaine d'activité UNERA > ACT-2 Bio-industries, biotechnologies
Code ID:1073
Déposé le :29 Mai 2006

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