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Mechanism of Antitermination by NusG-like Proteins and the Role of RNAP Conformational Mobility in Transcription CycleSevostiyanova, Anastasia K. 22 October 2010 (has links)
No description available.
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Defining the hierarchical regulation of BMP enhancers in early Drosophila developmentPinheiro, Marco January 2018 (has links)
Higher-order regulatory interactions between enhancer elements and target gene promoters have been implicated in the coordination and regulation of transcription in a spatio-temporal manner. Within development, the graded activity of enhancers controls transcriptional programs necessary to establish cell fates and tissue patterning. How enhancer promoter interactions form and dynamically change throughout development remains largely unknown. The aim of this thesis is to further characterise BMP enhancers during development. Using ChIP data, an enrichment of architectural binding proteins (ABPs) with enhancers regulated by the BMP pathway was identified. Analysis of chromatin signatures revealed a correlation with the active histone marks, H3K27ac and H3K36me3, over BMP enhancers enriched for the ABP BEAF32. BEAF32 mutants show disrupted expression of BMP target genes and altered tissue fates defined by the BMP pathway. Consequently, the role of BEAF32 genome-wide was considered, revealing interactions with factors associated with enhancers and promoters, in addition to a correlation with RNA Polymerase II (RNAPII) pausing at promoter regions. This suggests a possible role for BEAF32 in bridging enhancer promoter interactions and releasing paused RNAPII. Based on the prevalence of BEAF32 at some enhancer sites and interaction with CBP, eRNAs were identified within the Drosophila embryo, utilising available GRO-seq data and GroHMM. eRNA expression correlates to accessible enhancer states regardless of chromatin composition, with transcribed enhancers revealing interactions with active promoters, supporting correlations to transcriptional activation. Chromatin architecture of BMP targets were lastly considered using Capture-C against BMP regulated promoters, revealing multiple regulatory interactions including contacts with enhancers regulated along the Dorso-ventral (DV) axis and additional BMP promoters, with dynamic interactions between enhancers and promoters. Overall the presented data suggest that BMP promoters are dynamically regulated by distal enhancers, with a plausible role for BEAF32 in mediating enhancer promoter interactions, to co-ordinate transcription programs used to pattern dorsal tissues in the Drosophila embryo.
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Promoting Bacterial Synthesis of Oligo-prolines by Modifying Elongation Factor P Post-translationallyRajkovic, Andrei January 2016 (has links)
No description available.
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The Effect of Pause Duration on Intelligibility of Non-Native Spontaneous Oral DiscourseLege, Ryan Frederick 01 December 2012 (has links) (PDF)
Pausing is a natural part of human speech. Pausing is used to segment speech, negotiate meaning, and allow for breathing. In oral speech, pausing, along with other suprasegmental features, plays a critical role in creating meaning as comprehensible speech is seen as a goal for language learners around the world. In order to be comprehensible, language learners need to learn to pause correctly in their speaking. Though this notion is widely accepted by applied linguists and many language teachers, the effect of pausing on intelligibility of spontaneous oral discourse has not been established by empirical data. This study isolates pause duration in spontaneous oral discourse in order to establish its connection to the intelligibility of non-native speech. In this study, North American undergraduate students' reactions to non-native pause duration in spontaneous oral discourse were examined. The task involved measuring the NESs' processing, comprehension, and evaluation of three different versions of an international teaching assistant's presentation: One with unmodified pause duration, one with pause duration shortened by 50%, and a third passage with pause duration lengthened by 50%. Results showed a positive correlation between pause duration and number of listeners able to identify main ideas. Finally, listener reaction was measurably more positive to the unmodified passage than to the passages with lengthened or shortened pauses.
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Determining the mechanism of elongation factor P -dependent regulation of gene expressionElgamal, Sara January 2015 (has links)
No description available.
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Transcriptional and epigenetic control of gene expression in embryo developmentBoija, Ann January 2016 (has links)
During cell specification, temporal and spatially restricted gene expression programs are set up, forming different cell types and ultimately a multicellular organism. In this thesis, we have studied the molecular mechanisms by which sequence specific transcription factors and coactivators regulate RNA polymerase II (Pol II) transcription to establish specific gene expression programs and what epigenetic patterns that follows. We found that the transcription factor Dorsal is responsible for establishing discrete epigenetic patterns in the presumptive mesoderm, neuroectoderm and dorsal ectoderm, during early Drosophila embryo development. In addition, these different chromatin states can be linked to distinct modes of Pol II regulation. Our results provide novel insights into how gene regulatory networks form an epigenetic landscape and how their coordinated actions specify cell identity. CBP/p300 is a widely used co-activator and histone acetyltransferase (HAT) involved in transcriptional activation. We discovered that CBP occupies the genome preferentially together with Dorsal, and has a specific role during development in coordinating the dorsal-ventral axis of the Drosophila embryo. While CBP generally correlates with gene activation we also found CBP in H3K27me3 repressed chromatin. Previous studies have shown that CBP has an important role at transcriptional enhancers. We provide evidence that the regulatory role of CBP does not stop at enhancers, but is extended to many genomic regions. CBP binds to insulators and regulates their activity by acetylating histones to prevent spreading of H3K27me3. We further discovered that CBP has a direct regulatory role at promoters. Using a highly potent CBP inhibitor in combination with ChIP and PRO-seq we found that CBP regulates promoter proximal pausing of Pol II. CBP promotes Pol II recruitment to promoters via a direct interaction with TFIIB, and promotes transcriptional elongation by acetylating the first nucleosome. CBP is regulating Pol II activity of nearly all expressed genes, however, either recruitment or release of Pol II is the rate-limiting step affected by CBP. Taken together, these results reveal mechanistic insights into cell specification and transcriptional control during development. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.</p><p> </p>
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Regulation of transcription : structural studies of an RNA polymerase elongation complex bound to transcription factor NusA / Régulation de la transcription : études structurales du complexe d’élongation de l'ARN polymérase lié au facteur de transcription NusAGuo, Xieyang 04 September 2018 (has links)
La pause transcriptionnelle marquée par les ARN polymérases (RNAP) est un mécanisme clé pour réguler l'expression des gènes dans tous les règnes de la vie et est une condition préalable à la terminaison de la transcription. Le facteur de transcription bactérien essentiel NusA stimule à la fois la pause et la terminaison de la transcription, jouant ainsi un rôle central. Ici, je présente des reconstructions par cryo-microscopie électronique (cryo-EM) à une seule particule de NusA lié à des complexes d'élongation en présence et en absence d’ARN en épingle à cheveux dans le canal de sortie de l'ARN. Les structures révèlent quatre interactions entre NusA et RNAP qui suggèrent comment NusA stimule le repliement de l’ARN, la pause et la terminaison de la transcription. Un intermédiaire de translocation asymétrique de l'ARN et de l'ADN convertit le site actif de l'enzyme en un état inactif, fournissant une explication structurelle pour l'inhibition de la catalyse. La comparaison de RNAP à différentes étapes de la mise en pause donne un aperçu de la nature dynamique du processus et du rôle de NusA en tant que facteur de régulation. / Transcriptional pausing by RNA polymerases (RNAPs) is a key mechanism to regulate gene expression in all kingdoms of life and is a prerequisite for transcription termination. The essential bacterial transcription factor NusA stimulates both pausing and termination of transcription, thus playing a central role. Here, I present single-particle electron cryo-microscopy (cryo-EM) reconstructions of NusA bound to paused elongation complexes with and without a pause-enhancing hairpin in the RNA exit channel. The structures reveal four interactions between NusA and RNAP that suggest how NusA stimulates RNA folding, pausing, and termination. An asymmetric translocation intermediate of RNA and DNA converts the active site of the enzyme into an inactive state, providing a structural explanation for the inhibition of catalysis. Comparing RNAP at different stages of pausing provides insights on the dynamic nature of the process and the role of NusA as a regulatory factor.
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Low-temperature pausing : an alternative short-term preservation method for use in cell therapiesRobinson, Nathalie J. January 2016 (has links)
With encouraging advancements in cell therapies, there is a requirement for an effective short-term cell preservation method, enabling time for quality assurance testing and transport to their clinical destination. This project aims to pause cells at ambient temperatures, whilst maintaining viability and function post-preservation. Ambient cell preservation bypasses ice crystal exposure and toxic solute concentrations experienced with cryogenic storage. Storage in ambient conditions also avoids use of toxic cryoprotectants and aims to greatly reduce costs and reliability on specialist machinery. Early work used HOS TE85 cells (derived from an osteosarcoma) as a model. When atmospheric factors were controlled, HOS TE85 cells demonstrated effective recovery in terms of morphology, membrane integrity (viability >90%) and fold growth expansion when paused at ambient temperature for up to 144 hours. Without atmospheric control, addition of the buffering agent HEPES (25mM) to cell medium was required to keep viability above 70%, as well as to maintain yield and continual passage following 144 hours pausing. The pausing potential of therapeutically relevant human mesenchymal stem cells (hMSCs) from three individual donors (M2, M3 and M4) was tested by keeping cells in suspension for up to 72 hours. Using standard medium with the addition of 25mM HEPES, average membrane integrity was maintained above 70%. Following pausing for between 24 72 hours, hMSC attachment efficiency, immunophenotype and tri-lineage differentiation capacity (osteogenesis, adipogenesis and chondrogenesis) remained similar to non-paused cells. Apart from a short lag phase on the first passage, hMSC fold growth expansion level was consistent with the control for all three donors over 3 x 6 day passages. The colony forming unit (CFU) efficiency of paused cells was significantly reduced when compared with non-paused M2 and M4 lines, whilst M3 retained a similar CFU efficiency to its non-paused counterpart. On return to normal culture conditions, hMSCs had comparable metabolic activity rates with non-paused cells for up to 9 hours. Stable pH is vital during pausing and additional antioxidants or apoptotic inhibiters may be required to keep average viability well-above the 70% threshold, set by the US Food and Drug Administration. Collectively, results have been encouraging and show potential for the movement towards using ambient temperature preservation as an option for the short-term storage and transport of cells for therapy.
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Etude de la localisation à grande échelle de la machinerie de transcription de classe III, et de sa relation avec le facteur de transcription TFIIS dans les cellules souches embryonnaires de souris / Genomic binding of Pol III transcription machinery and relationship with TFIIS transcription factor distribution in mouse embryonic stem cellsCarriere, Lucie 29 September 2011 (has links)
Chez les eucaryotes, l’ARN polymérase (RNAP) III transcrit les ARN de transferts, l’ARN ribosomique 5S, et plusieurs douzaines d’autres ARNs non traduits. Le génome des mammifères contient plusieurs milliers d’éléments répétés, les SINEs. In vitro, leur transcription dépend de la RNAP III. Le taux de transcription de la RNAP III détermine la croissance et la prolifération cellulaires, sa dérégulation a été associée à de nombreux cancers. Afin de caractériser la distribution sur l’ensemble du génome de la RNAP III et de ses facteurs de transcription TFIIIB et TFIIIC, nous avons développé un protocole très spécifique de ChIP-seq en tandem. Nous avons déterminé l’ensemble des gènes liés par la RNAP III dans les cellules souches embryonnaires de souris. Cet ensemble est bien inférieur au nombre de gènes prédits dans le génome. Nous avons également observé la RNAP III et ses facteurs de transcription liés à 30 régions non annotées, seule une d’entre elles est conservée chez l’humain. Un très faible nombre de SINEs sur un demi-million prédits est associé à la RNAP III. Notre étude révèle de nombreux sites liés uniquement par TFIIIC, nommés « extra-TFIIIC loci », ETC chez la levure. Ces sites sont associés à la protéine CTCF, et à la cohésine. La cohésine occupe les sites liés par CTCF, et contribue à la formation de boucles ADN, associées à la répression ou à l’activation de l’expression des gènes. Ces données suggèrent que TFIIIC peut jouer un rôle dans l’organisation de l’architecture chromosomique chez les souris. Nous avons également démontré que TCEA1, l’isoforme ubiquitaire de TFIIS, le facteur d’élongation de la RNAP II, est associée aux gènes actifs de classe III. Ceci suggére que TFIIS est un facteur de transcription de classe III. Finalement, la distribution de TFIIS aux gènes de classe II indique que le recrutement de TFIIS n’est pas suffisant pour contrôler la transition de la RNAP II pausée en 5’ des gènes en élongation. / In eukaryotes, RNA polymerase (RNAP) III transcribes the tRNAs, the 5S ribosomal RNA and a half a dozen known untranslated RNA. Mammalian genome contains several thousand of repeated elements, the Short interspersed repetitive elements (SINE). In vitro, they are transcribed by RNAP III. RNAP III transcription levels determine cell growth and proliferation and, importantly, its deregulation is associated with cancer. Looking at the genome-wide distribution of RNAP III and its transcription factors, TFIIIB and TFIIIC, we develop a highly specific tandem ChIP-sequencing method. We have determined the set of genes that are transcribed by RNAP III in mouse embryonic stem cells. We discovered that not all known class III genes were transcribed in ES cells. We also observed that RNAP III and its transcription factors were present at thirty unannotated sites on the mouse genome, only one of which was conserved in human. Only a couple of hundreds of SINEs out of more than half a million are associated with RNAP III in mouse ES cells. Our study reveals numerous ‘TFIIIC-only’ sites, called ETC for extra-TFIIIC loci in yeast. These sites are correlated with association of CTCF and the cohesin. Cohesin has been shown to occupy sites bound by CTCF and to contribute to DNA loop formation associated with gene repression or activation. This observation suggests that TFIIIC may play a role in chromosome organization in mouse. We also demonstrated that TCEA1, the ubiquitous isoform of TFIIS RNAP II elongation factor, is associated with active class III genes suggesting that TFIIS is a RNAP III transcription factor in mammals. Finally, the distribution of TFIIS on RNAP II-transcribed genes indicated that its recruitment does not control the transition of RNAP II paused at genes 5’ end into elongation.
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Caractérisation du domaine C-terminal de l'ARN polymérase II et de la phosphatase Glc7 dans la terminaison transcriptionnelle chez Saccharomyces cerevisiaeCollin, Pierre 12 1900 (has links)
No description available.
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