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Evolution of the ribosomal common coreBernier, Chad R. 07 January 2016 (has links)
Understanding the origin of life requires understanding the origin of translation, which in turn, requires understanding the origin of the ribosome. Ribosomes are complex structures consisting of hundreds of thousands of atoms. Here, we describe how we organized ribosomal structures and information into a broad database, RiboZones. We also describe a new visualization web app, RiboVision.
RiboZones and RiboVision are productivity tools that lower the learning curve for ribosomal research. RiboZones makes the ribosome more accessible. RiboVision especially helps create beautiful publication ready figures in a fraction of the labor and time previously required. It is only through the creation of RiboZones and RiboVision through which the rest of this dissertation became feasible.
We constructed a high-quality sequence alignment of ribosomal sequences for both the LSU and the SSU rRNA. Each ribosomal sequence is complete, allowing detailed, low background statistics to be computed. The sequence alignment broadly samples the tree of life according to available data. The alignment was adjusted for maximum agreement with 3D superimpositions of multiple ribosomal structures.
We defined a nucleotide-level definition of the common core of the ribosome, as the RNA that is present in 95% of the sequences in our alignment. Multiple versions of the common core were created, including the universal common core, the prokaryotic common core, and domain specific common cores. The definition allows statistics to be computed for various use-cases. For example, with RiboVision visualization technology, it is possible to see which helices are optional, in which of the three domains of life, and what the minimum helical length is for each helix.
We discovered that ribosomal RNA grows mostly by helix extension and helix insertion. When a helix is inserted, it minimally perturbs the underlying helix. We call this pattern ‘insertion fingerprints’. Insertion fingerprints are found throughout the common core and the eukaryotic expansion segments.
Insertion fingerprints were used to divide the ribosomal RNA into units called ancestral expansion segments (AES’s). AES’s make ideal structural, functional, and evolutionary units. The AES’s are arranged into the first complete experimentally testable model of ribosomal evolution. The model can be refined over time as new information is discovered.
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Understanding the role of assembly factors in 30S subunit biogenesis / The role of assembly factors in 30S subunit biogenesisThurlow, Brett Thomas January 2016 (has links)
Our understanding regarding the function of YjeQ, RbfA, RimM and Era in ribosome biogenesis has been derived in part from the study of immature 30S particles that accumulate in bacteria strains lacking one of these factors. However, their mechanistic details are still unknown. Here, we demonstrate that the 30SΔyjeQ and 30SΔrimM immature particles are not dead-end products of assembly, but progress into mature 30S subunits. Mass spectrometry analysis revealed that in vivo the occupancy level of these factors in these immature 30S particles is below 10% and that the concentration of factors does not increase when immature particles accumulate in cells. Analysis of the binding interactions of these assembly factors with mature 30S subunits and the immature particles demonstrated that YjeQ and Era bind to the mature 30S subunit with high affinity, however binding of these factors to the immature particles and of RimM and RbfA to mature or immature particles is weak. This indicates that binding of the assembly factors to the immature particles is not occurring at physiological concentrations. These results suggest that in the absence of these factors, the immature particles evolve into a thermodynamically stable intermediate that exhibits low affinity for the assembly factors and that the true substrates of YjeQ, RbfA, RimM and Era are immature particles that precede the ribosomal particles accumulating in the knockouts strains. We also developed an Era-depletion and ΔrbfA strain, which exhibited slow-growth, cold-sensitivity and an aberrant ribosome profile, which are all characteristic of ribosome assembly defects. Cryo-EM structural analysis of the 30SEra-depleted particles revealed that multiple classes at various stages in the assembly process accumulate upon depletion of Era, suggesting that Era may have a global effect on biogenesis. Ultimately, this thesis provides new insights into the nature of 30S particles that accumulate during assembly factor perturbation and advances our understanding of ribosome biogenesis as a whole. / Dissertation / Doctor of Philosophy (PhD) / One of the most fundamental processes in all living cells is the synthesis of proteins by the ribosome. The ribosome is a massive macromolecular complex that consists of both proteins and RNA, which must be manufactured from its individual components before it can perform its function. There is a myriad of protein factors that assist in the assembly of ribosomes to ensure that biogenesis proceeds rapidly and efficiently. The purpose of this thesis was to gain a better understanding of how the assembly factors YjeQ, Era, RbfA and RimM work by studying the intermediates that accumulate when they are removed or depleted from the cell. Specifically, the fate, binding interactions and structure of the immature particles that accumulate in the assembly factor knockout or depletion strains were investigated. The work here brings new insights into the nature of these immature ribosomal particles and the maturation reactions catalyzed by these factors.
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Le ribosome au cours de l'érythropoïèse / Ribosome during erythropoiesisRaimbault, Anna 22 November 2016 (has links)
La biogenèse du ribosome est un processus indispensable à la prolifération cellulaire car elle permet la synthèse protéique assurant la croissance avant la division cellulaire. Les ribosomopathies telles que le syndrome myélodysplasique 5q- et l’anémie de Blackfan-Diamond sont dues respectivement à une mutation d’un gène codant une protéine ribosomique (RP) et à l’haploinsuffisance en RPS14, RP de la petite sous-unité du ribosome. Les patients atteints de l’une de ces ribosomopathies présentent un défaut de l’érythropoïèse suggérant que celle-ci est particulièrement dépendante du ribosome. L’érythropoïèse est le processus qui permet la formation de globules rouges à partir de cellules souches hématopoïétiques et consiste en différents stades de différenciation appelés érythroblastes. C’est dans ce contexte que je me suis intéressée au ribosome au cours de l’érythropoïèse. Dans un premier temps, nous avons caractérisé la biogenèse du ribosome dans des cellules érythroïdes primaires humaines et murines. Pour cela nous avons adapté une technique de SILAC pulsé et mis au point la ribomique, technique de protéomique permettant l’analyse de la biogenèse du ribosome dans des échantillons de cellules primaires basée sur l’identification presque exhaustive des protéines ribosomiques. À l’aide de la ribomique et par d’autres techniques, nous avons mis en évidence une diminution de la biogenèse du ribosome après le stade érythroblaste basophile. Nous avons également montré que cette biogenèse du ribosome est en partie sous le contrôle de la voie mTORC1 régulée par les deux cytokines fondamentales de l’érythropoïèse : le Stem Cell Factor (SCF) et l’érythropoïétine (EPO). L’expression par l’érythroblaste des récepteurs des deux cytokines permet une biogenèse du ribosome optimale. L’inhibition de la biogenèse du ribosome par le CX-5461, inhibiteur spécifique de l’ARN polymérase I, ou par la rapamycine, inhibiteur de mTORC1, entraîne une accélération de la différenciation érythroïde soulignant un rôle de la biogenèse du ribosome au cours de l’érythropoïèse. L’inhibition de la voie mTORC1 modifie l’ordre de clivage de l’ARNr, reflet d’une modification de sa maturation. Les expériences de ribomique dans les érythroblastes humains ont également permis de mettre en évidence la présence de paralogues de RP et la sous-représentation de certaines RPs au sein des ribosomes suggérant une hétérogénéité des ribosomes dans les érythroblastes humains. Parallèlement, un modèle mimant le syndrome 5q- a été développé par une approche shRPS14 dans une lignée humaine érythroleucémique dépendante de l’EPO. L’inhibition de RPS14 entraîne un défaut de biogenèse de la sous-unité 40S du ribosome aboutissant à une diminution des ribosomes entiers formés et une diminution de la traduction globale. Cependant une traduction est maintenue. Le défaut de biogenèse de la sous-unité 40S entraîne une augmentation de la quantité de c-KIT, récepteur du SCF et une diminution de la quantité de GATA1, facteur de transcription spécifique de l’érythropoïèse. Nous avons mis en évidence que la diminution de GATA1 est due à une diminution de sa traduction tandis que la traduction d’autres protéines est conservée dans ce contexte d’altération de la biogenèse du ribosome. Nous avons ensuite réalisé une analyse des transcrits présents dans les fractions polysomales correspondants à la traduction la plus efficace. Nous avons montré grâce à ce traductome que les propriétés thermodynamiques des parties 5’ et 3’UTR des ARNm modulent leur traduction dans le contexte d’inhibition de RPS14. Ces données suggèrent que l’altération de la biogenèse du ribosome peut aboutir à une modification du programme traductionnel. Ce travail montre que la biogenèse du ribosome diminue au cours de l’érythropoïèse et participe à la différenciation érythroïde. La voie mTORC1 participe au contrôle de cette biogenèse. / Ribosome biogenesis is a key event allowing cell growth before division. Defective RB recognized in ribosomopathyinherited Diamond-Blackfan anemia and 5q- syndrom. In this study, we aimed at investigating the regulatory role of RB during the erythroid precursor maturation which is characterized by a cell size reduction during 2 to 3 rapid cell divisions. We used two in vitro systemsé of expansion and differentiation of erythroblasts (E.) derived of immature hematopoietic progenitors from human mobilized peripheral blood or mouse fetal liver. The expansion step is supported by the Stem Cell Factor (SCF) and the second step depends on erythropoietin (EPO). The structure of the nucleolus was studied by electron microscopy. Compared to immature proerythroblasts (proE), a dramatic size reduction and change in nucleolar structure (ie. the disappearance of fibrillar and dense fibrillar components) is observed at the stage of mature polychromatophilic E. suggesting a loss of functionality. RB was measured by a pulsed SILAC (Stable Isotopic Labeling by Amino acids in Culture cell) proteomic assay that quantified the incorporation of newly synthesized ribosomal proteins in the ribosome. Both in mouse and human models, immature proE expanded upon SCF and EPO demonstrate a maximal RB with a renewal rate of 60% and 50% every 14h and 24h, respectively. By contrast, RB rapidly interrupted with the disappearance of proE and basophilic E after the switch to EPO alone. Consistently, the quantities of ribosomal RNA (rRNA) 45S precursor estimated by qPCR are maximal in proE and almost null in orthochromatophilic E. Inhibition of RB at proE stage by RNApol I specific inhibitor (CX-5461) accelerates the onset of terminal erythroid differentiation suggesting that RB is a rate limiting factor for final maturation. We then hypothesize that degree of signaling intensity in response to SCF and EPO may control the level of RB. To address this question, we investigated the mTORC1 (mechanistic Target Of Rapamycin Complex 1) pathway which is directly involved in RB through its substrate p70S6Kinase. Activation of P-p70S6Kinase and P-Rps6, as well as ribosome renewal, are twice more elevated in response to SCF and EPO than to EPO alone. Furthermore, inhibition of mTORC1/p70S6K/Rps6 pathway by rapamycin disrupts RB and leads to an acceleration of terminal erythroid differentiation.This study demonstrates that the collapse of RB promotes erythroid cell terminal maturation and shows the regulatory role of mTORC1 pathway on RB during erythropoiesis.
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Structural studies of a 5S RNA transcription factor and of the 5S RNA promoterMiller, Jonathan January 1987 (has links)
No description available.
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The molecular genetics of endocytosis and growth control in fission yeastHume, Alistair N. January 2000 (has links)
No description available.
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Studies on DNA sequences directing ribosomal transcription in Xenopus laevisSmith, D. R. January 1987 (has links)
No description available.
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Gene sequences encoding ribosome-inactivating proteins from soapwort (Saponaria officinalis L.)Fordham-Skelton, Anthony Paul January 1991 (has links)
Ribosome-inactivating proteins (RIPs) are found in a wide variety of plant species. They possess an RNA N-glycosidase activity whereby the removal of a specific adenine residue from 28 S RNA renders a eukaryotic ribosome inactive. Type II RIPS contain both an active polypeptide and a sugar-binding polypeptide. Type I RIPs are composed of a single polypeptide functionally homologous to the active type II polypeptide. This thesis describes studies of the gene sequences of RIPs representative of each class: Ricin, a type II RIP from the castor oil plant (Ricinus communis h.), and saporin, a type I RIP from soapwort (Saponaria officinalis L.). Two ricin gene sequences were isolated from a Ricinus genomic library and partially characterised. One gene was a badly damaged ricin-like pseudogene whilst the other was shown to encode an active polypeptide. A second ricin sequence encoding an active polypeptide was isolated using Polymerase Chain Reaction (PGR) DNA amplification. The specificity of PGR amplification was investigated using the ricin and related agglutinin gene sequences. Partial amino acid sequence data derived from protein sequencing of saporin-6 was used to synthesise degenerate inosine-containing oligonucleotides. These directed the PGR amplification of part of the saporin coding sequence from genomic DNA. The product was used as a saporin-specific hybridisation probe. Southern analysis of Saponaria genomic DNA indicated that saporin sequences comprised a small multigene family. Three independent saporin containing genomic clones were isolated from a Saponaria genomic library. Two clones were truncated whilst the third contained a complete saporin coding sequence. The saporin and ricin coding sequences were expressed in vitro and shown to inhibit protein synthesis. Aniline cleavage assays of ribosomal RNA extracted from ribosomes exposed to the products of the RIP coding sequences were carried out. These indicated that the polypeptides encoded by the RIP gene sequences had specific RNA N-glycosidase activity.
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Ribosome Processivity and Co-translational Protein FoldingThommen, Michael Sebastian 03 November 2015 (has links)
No description available.
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Mutational analysis of structure - function interactions within selected sites on the Escherichia coli ribosomeBelotserkovsky, Jaroslav Michailovich 08 February 2006 (has links)
Master of Science - Adult Education / Mutations were sought in Escherichia coli ribosomal RNA and ribosomal proteins
that confer dependence to the antibiotic streptomycin, using both newly available as well as well-established genetic systems. I found that a classical ribosomal mutant, Sm-D3, was streptomycin dependent and had an additional mutation in another ribosomal component – protein L7/L12. The double mutant had an 8-fold lower streptomycin requirement as compared to Sm-D3 with a wild-type rplL. This supported a functional involvement of L7/L12 in the decoding center of the ribosome.
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Úloha translačního iniciačního faktoru 3 v terminaci translace. / The role of translation initiation factor 3 (eIF3) in translation termination.Beznosková, Petra January 2016 (has links)
Protein synthesis is a tightly regulated process of gene expression. Each gene has its start and its stop, which is determined by one of the three stop codons. Many recent articles describe ribosomes that purposely bypass stops on specific mRNAs to extend the nascent polypeptide to alter its properties. It is called programmed stop codon readthrough. Since over 15% of human genetic diseases are caused by so called premature termination codons (PTC) that halt translation and produce truncated proteins, this mechanism has a great potential implication in medical research. Numerous labs search for non-toxic drugs specifically increasing readthrough at PTCs; however, the success of this effort requires identification and understanding of all factors that are involved in this process. Here, we present one such factor eukaryotic initiation factor 3 (eIF3) and describe its ability to induce readthrough on stop codons in termination non-favorable context during programmed readthrough and also the consequences of its action on translation regulation. We additionally analyzed which near-cognate (nc) tRNAs are incorporated at UGA stop codons depending on the nucleotide that immediately follows them (so called +4 base). This way we established new rules for stop codon decoding and identified so called...
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