Acknowledgments
Résumé
Abstract
1. Introduction
1.1. SRP cycle
1.2. Structure and functions
1.2.1. The different subunits of mammalian SRP
1.2.1.1. SRP RNA
1.2.1.2. SRP proteins
1.2.1.3. SRP receptor
1.3. Elongation arrest activity
1.3.1. Functional description
1.3.2. Structure of the Alu domain
1.3.3. Aim of the study
2. Results
2.1. SRP14
2.1.1. SRP14 carboxyl-terminus
2.1.2. SRP14 internal loop
2.1.3. Summary
2.2. SRP9
2.2.1 Summary
2.3. SRP RNA
2.3.1. SRP9/14 binding to mutated SRP RNAs
2.3.1.1. Mutations introduced in the RNA sequence based on biochemical data
2.3.1.2. Mutations based on the crystal structure
2.3.1.3. Electromobility shift assay
2.3.2. Functional analysis of particles containing mutated RNAs
2.3.2.1. The elongation arrest activity observed with the mutated particles is not due to a reduced SRP9/14 binding
2.3.3. Summary
3. Discussion
3.1. Alu SRP proteins
3.2. Alu SRP RNA
3.3. Model for elongation arrest activity
4. Materials and methods
4.1. Mutated RNAs genesis
4.2. Nomenclature used for the mutated RNAs
4.3. Large scale transcription
4.4. Protein purification
4.4.1. Human SRP9/14
4.4.2. Human SRP9/14K95
4.4.3. Human SRP9K41A/14 and SRP9K41R/14
4.4.4. Human SRP9/14sloop
4.4.5. Human SRP9/14-10Ct purification
4.4.6. SRP54 purification
4.4.7. SRP
f
19 purification
4.5. SRP reconstitutions
4.5.1. h9/14K95
4.5.2. hSRP9/14K95 competition in reconstitution
4.5.3. h9/14sloop and h9/14-10Ct
4.5.4. SRP9K41A/SRP14, SRP9K41R/SRP14
4.5.5. Reconstitution with mutated SRP RNAs
4.5.6. hSRP9/14 titration in 2L2 reconstitution
4.5.7. mutated RNA titration in reconstitution
4.6. RNA binding assay
4.7. Hydroxyl radical cleavage reaction
4.8. Electromobility shift assay
4.8.1. Native gels
4.8.2. Kd measure
4.9. Computer programs
Bibliography
Publication