No description available.

No description available.

La thèse présentée est centrée sur des mesures de force appliquées à des ARN seuls ou en interaction avec une protéine. L'un des plus grands défis posé par l'ARN provient de sa structure, notamment parce que les interactions de ses bases ne se limitent pas aux paires de type Watson-Crick et que les interactions tertiaires sont courantes. Les mesures de force donnent des informations complémentaires aux mesures classiques, car elles permettent de sonder directement les interactions complexes de l'ARN et fournissent des paramètres thermodynamiques inaccessibles autrement. L'étude de l'interaction protéine-ARN par mesure de force permet également d'accéder à des caractéristiques que les autres techniques ne peuvent pas offrir. Néanmoins avant d'atteindre les promesses de ce type de mesure, il faut concevoir un montage adapté et optimisé. Une première partie de la thèse a été dédiée à la conception d'une double pince optique permettant l'étude des ARN avec une résolution proche de la paire de base, en implantant notamment des améliorations techniques jusque là absentes de la littérature. La suite de la thèse s'est attachée à des études biologiques. Nous avons commencé par sonder l'interaction de la protéine ribosomique L20 avec son ARN cible dans le ribosome. Nous avons montré que cette protéine très importante dans les premiers stades de la formation du ribosome, stabilise fortement son ARN cible. Nous avons également déterminé son site de fixation à trois bases près. Nous avons ensuite débuté une étude sur des ARN synthétisés pour l'étude d'une hélicase à motif DEAD en caractérisant leurs réponses à une sollicitation mécanique.

<p>RNA is the molecular workhorse of nature, capable of doing many cellular tasks, from genetic data storage and regulation, to enzymatic synthesis--even to the point of self-catalyzing its own replication. While RNA can act as a catalyst on its own, as in the hammerhead ribozyme, the added efficiency of proteins is often a necessity; the ribosome--the large ribozyme responsible for peptide chain formation, is aided by proteins which ensure correct assembly and structural stability. These complexes of RNA and proteins feature in many essential cellular processes, including the RISC silencing complex and in the spliceosome. Despite its enormous utility, structural determination of RNA is notoriously difficult--particularly in the backbone, since a nucleotide standardly has 12 torsion angles (including &#967;) and 12 non-hydrogen atoms, compared to 4 torsions (including &#967;1) and 4 non-H atoms in a typical amino acid. The abundance of backbone atoms, their conformational flexibility, and experimental resolution limitations often result in systematic errors that can have a significant impact on the interpretation. False trails due to structural errors can lead to significant loss of time and effort, especially with such high-profile complexes as the ribosome and the RISC complex. </p><p>My research has focused on harnessing the recently discovered ribosome structures and the Richardsons' RNA dataset to find trends in RNA backbone conformations and motifs that were then used to develop structural validation techniques and provide improved diagnosis and correction techniques for RNA backbone. Methods for fixing RNA structure have been developed for both NMR and X-ray crystallography. For NMR structures, a method for assigning RNA backbone structure based on NOE data was developed, leading to improved identification and building of RNA backbone conformation in NMR ensembles. For crystallography, our method of diagnosing the correct ribose pucker from clear observables allows reliable assessment of pucker in validation or refinement. Observed differences in bond-lengths, bond-angles, and dihedrals have been categorized by sugar pucker in the PHENIX refinement package. I have shown that this improves the refinement behavior of both pucker and geometry. </p><p>There have also been improvements in identifying structural motifs. Many previously identified structural motifs have now been defined in terms of backbone suitestrings, a series of 2-character code divisions of RNA backbone that show the best clustering of dihedral angle correlations. Combined with a BLAST-like alignment program called SuiteAlign, these suitestrings were quickly and easily identified in a number of structures, eventually leading to the discovery of multiple instances of T&#968;C-loop structures in the ribosome.</p><p>To facilitate error diagnosis and corrections in RNA-protein complexes, as well as to expand the knowledge base of the scientific community as a whole, a database of RNA-protein interaction motifs has been developed. This database is rooted in the quality-filtering, visualization, and analysis techniques of the Richardson lab, particularly those developed by Laura Murray specifically for RNA structures.</p><p>The consensus backbone conformers, pucker diagnosis, and all-atom contacts have been combined to develop first manual and then automated tools for RNA structure correction. I have applied all these techniques to improve the accuracy of a number of important RNA and RNA/protein complex structures.</p> / Dissertation

RelA is a guanosine tetraphosphate synthetase which catalyzes the production of (p)ppGpp during the stringent response in Escherichia coli. RelA consists of an N-terminus, which is responsible for the catalytic activity, and a C-terminus, which is thought to be involved in the regulation of RelA activity. Furthermore, the C-terminus has dimerization and ribosome binding ability. ‘RelA-2, which is a fragment of C-terminus, is a major domain responsible for dimerization and ribosome binding. In this study, it was demonstrated that combination of two mutations (C612G, D637R) in ‘RelA-2 significantly reduced the dimerization. This dimerization-defective mutant still bound to ribosomes both in vivo and in vitro, indicating that dimerization is not required for its ribosome binding and that the dimerizaton domain is separated from its ribosome binding domain. The overexpression of the dimerization-defective mutant in amino acid starved cells inhibited chromosome-encoded wild type RelA activity. As a result, the starved cells did not show a stringent response. This finding does not support the oligomerization model proposed by Gropp group. Previous studies in this laboratory have shown, and were confirmed here, that the overexpressed ‘RelA-3, another fragment of C-terminus, which is devoid of dimerization and ribosome binding ability, did not inhibit the RelA activity when cells are under amino acid starvation. This evidence supports the hypothesis that ribosome binding is somehow involved in the regulation of RelA activity. It was demonstrated in this study that RelA was localized to the 50S subunit in vivo by Western Blot analysis. This result confirmed a previous study showing that the 50S subunit had the enzymatic activity in vitro, but not the 30S subunit. However, an in vitro study using pure 50S and 30S ribosomal subunits for the binding experiments indicated that RelA mainly bound to the 30S subunit and weakly to the 50S subunit. A model has been proposed to explain the possible mechanism of ribosome association for RelA. The involvement of L11 and EF-G in the regulation of RelA activity was also investigated. Three residues (C38, G131, and G137) in L11 have been identified to be crucial for the regulation of RelA activity. Three residues (T89, L438, and G628) in EF-G have been identified to be involved in the regulation of RelA activity. These preliminary studies implicate that the regulation of RelA inside amino acid-starved E. coli is complex.

This thesis addresses different questions regarding the rate, efficiency, and accuracy of peptide bond formation with natural as well as unnatural amino acids: Which step is rate-limiting during peptide bond formation? How does the accuracy vary with different transfer RNAs (tRNAs) and codons and how is it relevant to the living cells? Does proofreading selection of codon reading occur in a single- or multi-step manner as theoretically suggested? How does the E. coli translation system discriminate unnatural amino acids? Based on that, how to improve the incorporation efficiencies of unnatural amino acids? Based on the study on pH dependence of peptide bond formation, we show that the rate of the chemistry of peptidyl transfer to aminoacyl-tRNA (AA-tRNA) Gly-tRNAGly or Pro-tRNAPro limits the rate of peptide bond formation at physiological pH 7.5, and this could possibly be true for peptidyl transfer to all natural AA-tRNAs at physiological condition. By studying the efficiency-accuracy trade-off for codon reading by seven AA-tRNA containing ternary complexes, we observe a large variation on the accuracy of initial codon selection and identify several error hot-spots. The maximal accuracy varied 400-fold from 200 to 84000 depending on the tRNA identity, the type and position of the mismatches. We also propose a proofreading mechanism that contains two irreversible steps in sequence. This could be highly relevant to the living cells in relation to maintaining both high accuracy and high efficiency in protein synthesis. Finally, we show that peptide bond formation with small and large non-N-alkylated L- unnatural amino acids proceed at rates similar to those with natural amino acids Phe and Ala on the ribosome. Interestingly, the large side chain of the bulky unnatural amino acid only weakens its binding for elongation factor Tu (EF-Tu) but not slows down peptidyl transfer on the ribosome. Our results also suggest that the efficiency of unnatural amino acid incorporation could be improved in general by increasing EF-Tu concentration, lowering the reaction temperature and / or using tRNA bodies with optimal affinities for EF-Tu in the translation system.

No description available.

The structural basis for the fidelity of translation was modeled using computational methods. The flexibility of tRNA was explored using molecular dynamics and making a database of all crystallographic structures for tRNA. The modes of flexibility were compared based upon several metrics. A method for fitting cryo-EM with crystallographic structures was developed (MdMD), and also, for finding pathways between cryo-EM states. Biasing methods in molecular dynamics were used to model the pathway for the proofreading step of ribosomal translation. Atomic models were made for the Pre- and Post- accommodation state of the ribosome. These results indicated a new hypothesis for the mechanism of proofreading. There was no evidence for an induced fit mechanism in the large or small subunit of the ribosome during this step. The tRNA has a differential deformation in the kink during decoding that is based upon whether the tRNA is cognate or near-cognate. This difference in stored energy affects the outcome of proofreading, and the simulations of this step show that the ribosome presents some barriers, which would reject tRNA with insufficient stored energy.

The current work reports on structural studies of ribonucleoprotein complexes, Escherichia coli and Thermomyces lanuginosus ribosomes, and Pariacoto virus (PaV) using molecular modeling. Molecular modeling is the integration and representation of the structural data of molecules as models. Integrating high-resolution crystal structures available for the E. coli ribosome and the cryo-EM density maps for the PRE- and POST- accommodation states of the translational cycle, I generated two all-atom models for the ribosome in two functional states of the cycle. A program for flexible fitting of the crystal structures into low-resolution maps, YUP.scx, was used to generate the models. Based on these models, we hypothesize that the kinking of the tRNA plays a major role in cognate tRNA selection during accommodation. Secondly, we proposed all-atom models for the eukaryotic ribosomal RNA. This is part of a collaboration between Joachim Frank, Andrej Sali, and our lab to generate an all-atom model for the eukaryotic ribosome based on a cryo-EM density map of T. lanuginosus available at 8.9Å resolution. Homology modeling and ab initio RNA modeling were used to generate the rRNA components. Finally, we propose a first-order model for a T=3, icosahedral, RNA virus called Pariacoto virus. We used the structure available from x-ray crystallography as the starting model and modeled all the unresolved RNA and protein residues. Only 35% of the total RNA genome and 88% of the protein were resolved in the crystal structure. The generated models for the virus helped us determine the location of the missing N-terminal protein tails. The models were used to propose a new viral assembly pathway for small RNA viruses. We propose that the basic N-terminal tails make contact with the RNA genome and neutralize the negative charges in RNA and subsequently collapse the RNA/protein complex into a mature virus. This process is reminiscent of DNA condensation by positively charged ions.

RNA is amazing. We found that without changing the backbone connectivity, RNA can maintain structural conservation in 3D via topology switches, at a single residue level. I developed a method of representing RNA structure in multiresolution, called the PBR approach (P stands for Phosphate; B stands for Base; R stands for Ribose). In this method, structural data is viewed through a series of resolutions from finest to coarsest. At a single nucleotide resolution (fine resolution), RNA is abstruse and elaborate with structural insertions/deletions, strand clips, and 3,2-switches. The compilation of structural deviations of RNA, called DevLS (Deviations of Local Structure), provides a new descriptive language of RNA structure, allowing one to systematize and investigate RNA structure. Using PBR analysis, a total of 103 tetraloops within the crystal structures of the 23s rRNA of H. marismortui and the 70s rRNA of T. thermophilus are found and classified. Combining them, I constructed a 'tetraloop family tree', using a tree formalism, to unify and re-define the tetraloop motif and to represent relationships between tetraloops, as grouped by DevLS. To date, structural alignment of very large RNAs remains challenge due to the large size, intricate backbone choreography, and tertiary interactions. To overcome these obstacles, I developed a concept of structural anchors along with a 'Divide and Conquer' strategy for performing superimposition of 23s rRNAs. The successful alignment and superimpositions of the 23s rRNAs of T. thermophilus and H. marismortui gives an overall RMSD of atomic positions of 1.2 Å, as utilized 73% of RNA backbone atoms (~ 2129 residues). By using principles of inorganic chemistry along with structural alignment technique as described above, a recurrent magnesium-binding motif in large RNAs is revealed. These magnesium-binding motifs play a critical role in the framework of the ribosomal PTC by their locations, topologies, and coordination geometries. Common features of Mg2+-mc's include direct phosphate chelation of two magnesium ions in the form of Mg2+(i)-(O1P-P-O2P)-Mg2+(j), phosphate groups of adjacent RNA residues as ligands of a given Mg2+, and undulated RNA surfaces with unpaired and unstacked bases.