Les ARNnc sont impliqués dans la régulation de l’expression des gènes via divers mécanismes. Ils adoptent des structures 3D composées à 70% de pb WC formant des hélices de types A liées entre elles par des jonctions modulaires ayant des caractéristiques géométriques spécifiques. Nous avons identifié un nouveau motif 3D d’ARN apparenté au kturn, le pk-turn. Le pk-turn, situé dans la RNase P bactérienne permet, comme le k-turn, la formation d’un angle de 60° entre les hélices P16 & P17 avec cependant des exigences de séquences et de structure différentes. Le 2nd ribozyme qui a focalisé mon attention est le LCrz observé dans l’intron siamois (GIR2/LCrz) identifié dans le pré-ARNr 18S de la petite sous unité du ribosome eucaryote du myxomycète D. iridis. LCrz catalyse une réaction de branchement, équivalente à la première étape de l’épissage par les introns de groupe II, dans un contexte structural proche des introns du groupe I. Nous avons résolu la structure cristallographique du LCrz à une résolution de 2.5Å révélant un repliement inattendu. Cette structure a été confirmée par des expériences de SAXS. Ce travail nous permet de souligner la relation entre structure et fonction dans l'évolution des ribozymes. / NcRNA represent most of primary transcripts RNA in higher eukaryotes and tune gene expression via diverse mechanisms. They adopt 3D structures composed at 70% by WC bp forming A-form helices linked by RNA motifs. We identified the pk-turn, a new RNA motif related to k-turns that allow for the formation of a bend of 60° between stems P16 and P17 from the bacterial RNaseP. Yet it features different sequence and structural requirements than k-turns. The 2nd ribozyme which got my attention is the LCrz inserted in GIR2, a group I intron. This twintron is observed in the pre-rRNA 18S of the small subunit of the eukaryoteD. iris. LCrz catalyzes a reaction equivalent to the first step of splicing by group II introns, but in a structural context related to group I introns. We solved the 2.5 Å crystal structure of the LCrz and confirmed the unexpected shape by means of SAXS experiments. This work emphasizes the relationship between structure and function in the evolution of ribozymes.

No description available.

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.

No description available.

In recent years, several interesting biological roles played by RNA have come to light. Apart from their known role in translation of genetic information from DNA to protein, they have been shown to act as enzymes as well as regulators of gene expression. Protein-RNA complexes are involved in regulating cellular processes like cell division, differentiation, growth, cell aging and death. A number of clinically important viruses have RNA as their genetic material. Defective RNA molecules have been linked to a number of human diseases. The ability of RNA to adopt stunningly complex three-dimensional structures aids in diverse functions like catalysis, metabolite sensing and transcriptional control. Several secondary structure motifs are observed in RNA, of which the double-helical RNA motif is ubiquitous and well characterized. Though DNA duplexes have been shown to be present in many polymorphic states, RNA duplexes are believed to exhibit conservatism. Early fibre diffraction analysis on molecular structures of natural and synthetically available oligo- and polynucleotides suggested that the double-helical structures of RNA might exist in two forms: A-form and A′-form. New improved crystallographic methods have contributed to the increased availability of atomic resolution structures of many biologically significant RNA molecules. With the available structural information, it is feasible to try and understand the contribution of the variations at the base pair, base-pair step and backbone torsion angle level to the overall structure of the RNA duplex. Further, the effect of protein binding on RNA structure has not been extensively analysed. These studies have not been investigated in greater detail due to the focus of the research community on understanding conformational changes in proteins when bound to RNA, and due to the lack of a significant number of solved RNA structures in both free and protein-bound state. While studies on the conformation of the DNA double-helical stem have moved beyond the dinucleotide step into tri-, tetra-, hexa- and octanucleotide levels, similar knowledge for RNA even at the dinucleotide step level is lacking. In this thesis, the results of detailed analyses to understand the contribution of the base sequence towards RNA conformational variability as well as the structural changes incurred upon protein binding are reported. Objectives The primary objective of this thesis is to understand the following through detailed analyses of all available high-resolution crystal structures of RNA. 1 Exploring sequence-dependent variations exhibited by dinucleotide steps formed by Watson-Crick (WC) base pairs in RNA duplexes. 2 Identifying sequence-dependent variations exhibited by dinucleotide steps containing non-Watson-Crick (NWC) base pairs in RNA duplexes. 3 Developing a web application for the generation of sequence-dependent non-uniform nucleic acid structures. 4 Investigating the relationship between base sequence and backbone torsion-angle preferences in RNA double helices followed by molecular dynamics simulation using various force fields, to check their ability to reproduce the above experimental findings. Chapter 1 gives an overview of the structural features and polymorphic states of RNA duplexes and the present understanding of the structural architecture of RNA, thereby laying the background to the studies carried out subsequently. The chapter also gives a brief description on the methodologies applied. Relevant methodologies and protocols are dealt with in detail in the respective chapters. Sequence-dependent base-pair step geometries in RNA duplexes A complete understanding of the conformational variability seen in duplex RNA molecules at the dinucleotide step level can aid in the understanding of their function. This work was carried out to derive geometric information using a non-redundant RNA crystal structure dataset and to understand the conformational features (base pair and base-pair step parameters) involving all Watson-Crick (WC) (Chapter 2) and non-Watson-Crick (NWC) base pairs (Chapter 3). The sequence-dependent variations exhibited by the base-pair steps in RNA duplexes are elaborated. Further, potential non-canonical hydrogen bond interactions in the steps are identified and their relationship with dinucleotide step geometry is discussed. Comparison of the features of dinucleotide steps between free and protein-bound RNA datasets suggest variations at the base-pair step level on protein binding, which are more pronounced in non-Watson-Crick base pair containing steps. Chapter 4 describes a web-server NUCGEN-Plus, developed for building and regeneration of curved and non-uniform DNA and RNA duplexes. The main algorithm is a modification of our earlier program NUCGEN that worked mainly for DNA. The WC step parameters and intra-base parameters for RNA were obtained from the work detailed in Chapter 2. The FORTRAN code and input sequence file format was modified. The program has two modules: a) Using the model-building module, the program can build duplex structures for a given input DNA/RNA sequence. Options are available for selecting various derived or user specified base-pair step parameters, and fibre diffraction parameters that can be used in the building process. The program can generate double-helical structures up to 2000 nucleotides in length. In addition, the program can calculate the curvature of the generated duplex at defined length scale. b) Using the regeneration module, double-helical structures of nucleic acids can be rebuilt from the existing solved structures. Further, variants of an existing structure can be generated by varying the input geometric parameters. The web-server has a user-friendly interface and is freely available in the public domain at: http://nucleix.mbu.iisc.ernet.in/nucgenplus/index.html Sequence dependence of backbone torsion angle conformers in RNA duplexes RNA molecules consist of covalently linked nucleotide units. Each of these units has six rigid torsional degrees of freedom (α, β, γ, δ, ε, and ζ) for the backbone and one (χ) around the glycosidic bond connecting the base to the ribose, thereby providing conformational flexibility. An understanding of the relationship between base sequence and structural variations along the backbone can help deduce the rationale for sequence conservation and also their functional importance. Chapter 5 describes in detail the torsion angle-dependent variations seen in dinucleotide steps of RNA duplex. A non-redundant, high resolution (≤2.5Å) crystal structure dataset was created. Base-specific preferences for the backbone and glycosidic torsion angles were observed. Non-A-form torsion angle conformers were found to have a greater prevalence in protein-bound duplexes. Further validation of the above observation was performed by analysing the RNA backbone conformers and the effect of protein binding, in the crystal structure of E. coli 70S ribosome. Chapter 5 further describes the molecular dynamics simulation studies carried out to understand the effect of force fields on the RNA backbone conformer preferences. A 33mer long duplex was simulated using seven different force fields available in AMBER and CHARMM program, each for 100 ns. Trajectory analyses suggest the presence of sequence-dependent torsion angle preferences. Torsion angle conformer distribution closer to that of crystal structures was observed in the system simulated using parmbsc0 force field. Molecular dynamics simulation studies of AU/AU base-pair step A unique geometric feature, unlike that in other purine-pyrimidine (RY) steps in the crystal dataset analysis, was reported for AU/AU step (see Chapter 2). Appendix 1 describes the work carried out to validate these features observed in the crystal structures using simulation studies. Additionally, the effect of nearest-neighbor base pairs on the AU/AU step geometry were examined. General Conclusion Overall, the findings of this thesis work suggest that RNA duplexes exhibit sequence-dependent structural variations and sample a large volume of the double-helical conformational space. Further, protein binding affects the local base-pair step geometry and backbone conformation.

In this work we use a graph-theoretic representation of secondary RNA structure found in the database RAG: RNA-As-Graphs. We model the bonding of two RNA secondary structures to form a larger structure with a graph operation called merge. The resulting data from each tree merge operation is summarized and represented by a vector. We use these vectors as input values for a neural network and train the network to recognize a tree as RNA-like or not based on the merge data vector. The network correctly assigned a high probability of RNA-likeness to trees identified as RNA-like in the RAG database, and a low probability of RNA-likeness to those classified as not RNA-like in the RAG database. We then used the neural network to predict the RNA-likeness of all the trees of order 9. The use of a graph operation to theoretically describe the bonding of secondary RNA is novel.

No description available.

Le ribozyme VS de Neurospora catalyse des réactions de clivage et de ligation d’un lien phosphodiester spécifique essentielles à son cycle de réplication. Il est formé de six régions hélicales (I à VI), qui se divisent en deux domaines, soit le substrat (SLI) et le domaine catalytique (tiges II à VI). Ce dernier comprend deux jonctions à trois voies qui permettent de reconnaître le substrat en tige-boucle de façon spécifique. Ce mode de reconnaissance unique pourrait être exploité pour cibler des ARN repliés pour diverses applications. Bien que le ribozyme VS ait été caractérisé biochimiquement de façon exhaustive, aucune structure à haute résolution du ribozyme complet n’a encore été publiée, ce qui limite la compréhension des mécanismes inhérents à son fonctionnement. Précédemment, une approche de divide-and-conquer a été initiée afin d’étudier la structure des sous-domaines importants du ribozyme VS par spectroscopie de résonance magnétique nucléaire (RMN) mais doit être complétée. Dans le cadre de cette thèse, les structures de la boucle A730 et des jonctions III-IV-V et II-III-VI ont été déterminées par spectroscopie RMN hétéronucléaire. De plus, une approche de spectroscopie RMN a été développée pour la localisation des ions divalents, tandis que diverses approches de marquage isotopique ont été implémentées pour l’étude d’ARN de plus grandes tailles. Les structures RMN de la boucle A730 et des deux jonctions à trois voies révèlent que ces sous-domaines sont bien définis, qu’ils sont formés de plusieurs éléments structuraux récurrents (U-turn, S-turn, triplets de bases et empilement coaxial) et qu’ils contiennent plusieurs sites de liaison de métaux. En outre, un modèle du site actif du ribozyme VS a été construit sur la base des similarités identifiées entre les sites actifs des ribozymes VS et hairpin. Dans l’ensemble, ces études contribuent de façon significative à la compréhension de l’architecture globale du ribozyme VS. De plus, elles permettront de construire un modèle à haute résolution du ribozyme VS tout en favorisant de futures études d’ingénierie. / The Neurospora VS ribozyme catalyzes the cleavage and the ligation of a specific phosphodiester bond, which is essential for its replication cycle. It is formed of six helical regions (I to VI) that are divided in two domains: the substrate (SLI) and the catalytic domain (stems II-VI). The latter contains two three-way junctions that allow recognition of the stem-loop substrate in a specific manner. This unique mode of substrate recognition could be exploited to target folded RNAs for diverse applications. Even though the VS ribozyme has been extensively characterized biochemically, no high-resolution structure of the complete ribozyme has been published yet and this limits our mechanistic understanding. A divide-and-conquer approach was previously initiated to study the structure of the important subdomains of the VS ribozyme by nuclear magnetic resonance (NMR), but this approach needs to be completed. In this thesis, the structures of the A730 loop, the III-IV-V junction and the II-III-VI junction were determined by heteronuclear NMR spectroscopy. Moreover, a unique NMR approach was developed for localizing divalent metal ions, whereas several isotope-labeling strategies were implemented to facilitate the study or large RNA molecules. The NMR structures of the A730 loop and the two three-way junctions reveal that these subdomains are well defined, that they are formed by several recurrent structural elements (U-turn and S-turn motifs, base triples and coaxial stacking) and that they contain several metal-binding sites. Interestingly, structural similarities were identified between the VS and hairpin ribozymes, which allowed the modeling of the VS ribozyme active site. In summary, these studies significantly contribute to a better understanding of the global architecture of the VS ribozyme. In addition, they will allow the construction of a high-resolution model of the complete VS ribozyme and facilitate future engineering studies.

No description available.