Dissertationen zum Thema „Interactions protein-RNA“
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1
Hahn, Daniela. „Brr2 RNA helicase and its protein and RNA interactions“. Thesis, University of Edinburgh, 2011. http://hdl.handle.net/1842/5775.
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The dynamic rearrangements of RNA and protein complexes and the fidelity of pre-mRNA splicing are governed by DExD/H-box ATPases. One of the spliceosomal ATPases, Brr2, is believed to facilitate conformational rearrangements during spliceosome activation and disassembly. It features an unusual architecture, with two consecutive helicase-cassettes, each comprising a helicase and a Sec63 domain. Only the N-terminal cassette exhibits catalytic activity. By contrast, the C-terminal half of Brr2 engages in protein interactions. Amongst interacting proteins are the Prp2 and Prp16 helicases. The work presented in this thesis aimed at studying and assigning functional relevance to the bipartite architecture of Brr2 and addressed the following questions: (1) What role does the catalytically inert C-terminal half play in Brr2 function, and why does it interact with other RNA helicases? (2) Which RNAs interact with the different parts of Brr2? (1) In a yeast two-hybrid screen novel brr2 mutant alleles were identified by virtue of abnormal protein interactions with Prp2 and Prp16. Phenotypic characterization showed that brr2 C-terminus mutants exhibit a splicing defect, demonstrating that an intact C-terminus is required for Brr2 function. ATPase/helicase deficient prp16 mutants suppress the interaction defect of brr2 alleles, possibly indicating an involvement of the Brr2 C-terminus in the regulation of interacting helicases. (2) Brr2-RNA interactions were identified by the CRAC approach (in vivo Crosslinking and analysis of cDNA). Physical separation of the N-terminal and C-terminal portions and their individual analyses indicate that only the N-terminus of Brr2 interacts with RNA. Brr2 cross-links in the U4 and U6 snRNAs suggest a step-wise dissociation of the U4/U6 duplex during catalytic activation of the spliceosome. Newly identified Brr2 cross-links in the U5 snRNA and in pre-mRNAs close to 3’ splice sites are supported by genetic analyses. A reduction of second step efficiency upon combining brr2 and U5 mutations suggests an involvement of Brr2 in the second step of splicing. An approach now described as CLASH (Cross-linking, Ligation and Sequencing of Hybrids) identified Brr2 associated chimeric sequencing reads. The inspection of chimeric U2-U2 sequences suggests a revised secondary structure for the U2 snRNA, which was confirmed by phylogenentic and mutational analyses. Taken together these findings underscore the functional distinction of the N- and C-terminal portions of Brr2 and add mechanistic relevance to its bipartite architecture. The catalytically active N-terminal helicase-cassette is required to establish RNA interactions and to provide helicase activity. Conversely, the C-terminal helicase-cassette functions solely as protein interaction domain, possibly exerting regulation on the activities of interacting helicases and Brr2 itself.
2
Chandran, V. „Structural and functional characterisation of the protein-protein and protein-RNA interactions in the RNA degradosome“. Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.597437.
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The C-terminal domain of RNase E is intrinsically unstructured, but small segments of 13 to 80 residues are predicted to have propensity for defined conformation and evidence presented here indicates that they function in nucleic acid binding and protein-protein interactions in the degradosome. Binding of two of these ordered regions to their predicted partners, enolase and PNPase, has been demonstrated using non-dissociating nano-flow mass spectrometry (MS). Binding of helicase to an arginine rich domain of RNase E (residues 628-843) has also been shown by MS and other approaches. The binding stoichiometry for the various degradosome components with minimal binding regions on RNase E is also provided by non-dissociating MS, and these data provide insight into the organisation of the degradosome. The crystal structure of E. coli enolase in complex with a synthetic peptide corresponding to the proposed recognition in the degradosome (RNase E residues 833-850) has been solved to 1.6Å resolution. The crystal structure reveals a 1:1 complex of the peptide with the enolase dimer. It is predicted that enolase and RNase E interact specifically and stably in many Gram-negative pathogens, with implications for a common mode of regulated turnover of targeted transcripts. An analysis of the metabolome data from wild type E. coli cells and the degradosome mutant strains grown normally and under phosphosugar stress signify the importance of coordinated RNA degradation by the organisation of a degradosome machinery in regulating cellular responses to physiological stress stimuli.
3
Batal, Rami. „RNA and protein interactions of the measles virus nucleocapsid protein“. Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=55437.
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We are interested in studying the RNA and protein binding activities of the measles virus (MV) NP. MV is one of the members of Paramyxoviridae, a family of non-segmented negative-stranded RNA viruses family. We have expressed the MV NP in procaryotic systems and by in vitro translation. We have created a number of carboxy-terminal deletions of NP to use in mapping the domains involved in RNA and protein binding. We have transcribed the 5$ sp prime$ end antigenome sequences (positive leader) in vitro. We have metabolically labeled viral and MV-infected cellular proteins. We have applied different RNA-protein and protein-protein binding assays in order to study the postulated binding. We were not able to unequivocally detect specific binding between NP and the RNA. However, we have observed significant binding between NP and each of three MV-specific proteins NP, P, and M. Furthermore, we have found that the carboxy terminus of NP is important in this binding. Deletions in that domain will abolish M binding, and further deletions towards the amino terminus will abolish P binding, and eventually NP binding. Successively larger carboxy-terminal deletions first abolish NP binding to M, then to P, and then eventually to itself.
4
Xu, Deming. „RNA and protein interactions in the yeast spliceosome“. Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape10/PQDD_0005/NQ41534.pdf.
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5
Turner, David Richard. „Protein-RNA interactions in tobacco mosaic virus assembly“. Thesis, University of Cambridge, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328799.
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6
Singh, Jagjit. „RNA-Protein Interactions in the U12-Dependent Spliceosome“. Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1484307043050366.
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7
Terribilini, Michael Joseph. „Computational analysis and prediction of protein-RNA interactions“. [Ames, Iowa : Iowa State University], 2008.
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8
Peters, Daniel. „Structural and biochemical investigation of protein-RNA interactions“. Thesis, University of York, 2014. http://etheses.whiterose.ac.uk/6784/.
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Non-coding RNAs (ncRNAs) are nucleic acids that do not code for protein. Rather, they have evolved highly specialised secondary structures and catalytic mechanisms that place them at the heart of regulating gene expression. The function of ncRNAs is often mediated or dependent on their interactions with RNA binding proteins. The study of both the structure and function of these proteins is crucial for understanding the biological role of the protein-RNA complexes. In this thesis, the structure and function of two RNA binding proteins: Lin28 and dihydrouridine synthase C (DusC) were investigated using X-ray crystallography and biophysical techniques. In both systems, the specific recognition of target molecules is important for function. The aim of the study was therefore to use structural and functional data to elucidate the molecular basis of these protein-RNA interactions. There are three main findings: (1) specific recognition of microRNAs by Lin28 is dependent on the interaction of the Zinc Knuckle domain of the protein with a 3’ GGAG motif; (2) non-specific, electrostatic interactions between the cold-shock domain of Lin28 and RNA suggest a transcriptome scanning mechanism for recognising Lin28 targets; and (3) modification of specific nucleotide positions within tRNA by DusC is dependent on the orientation in which the tRNA is bound, which is determined by minor changes in the protein structure. These findings have helped to elucidate the mechanisms, and hence biological functions, of these RNA binding proteins. Both proteins have been previously associated with cancer. Through greater understanding of the molecular basis of these protein-RNA interactions, the production of novel therapeutic agents can be informed, which can help to combat disease. This data will therefore aid future efforts to treat and prevent the cancers caused by the aberrant actions of these RNA binding proteins.
9
Ellis, Jonathan James. „Towards the prediction of protein-RNA interactions through protein structure analysis“. Thesis, University of Sussex, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.444117.
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10
Ribeiro, Diogo. „Discovery of the role of protein-RNA interactions in protein multifunctionality and cellular complexity“. Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0449/document.
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Au fil du temps, la vie a évolué pour produire des organismes remarquablement complexes. Pour faire face à cette complexité, les organismes ont développé une pléthore de mécanismes régulateurs. Par exemple, les mammifères transcrivent des milliers d'ARN longs non codants (ARNlnc), accroissant ainsi la capacité régulatrice de leurs cellules. Un concept émergent est que les ARNlnc peuvent servir d'échafaudages aux complexes protéiques, mais la prévalence de ce mécanisme n'a pas encore été démontrée. De plus, pour chaque ARN messager, plusieurs régions 3’ non traduites (3’UTRs) sont souvent présentes. Ces 3’UTRs pourraient réguler la fonction de la protéine en cours de traduction, en participant à la formation des complexes protéiques dans lesquels elle est impliquée. Néanmoins, la fréquence et l’importance ce mécanisme reste à aborder.Cette thèse a pour objectif de découvrir et comprendre systématiquement ces deux mécanismes de régulation méconnus. Concrètement, l'assemblage de complexes protéiques promus par les ARNlnc et les 3'UTRs est étudié avec des données d’interactions protéines-protéines et protéines-ARN à grande échelle. Ceci a permis (i) de prédire le rôle de plusieurs centaines d'ARNlnc comme molécules d'échafaudage pour plus de la moitié des complexes protéiques connus, ainsi que (ii) d’inférer plus d’un millier de complexes 3'UTR-protéines, dont certains cas pourraient réguler post-traductionnellement des protéines moonlighting aux fonctions multiples et distinctes. Ces résultats indiquent qu'une proportion élevée d'ARNlnc et de 3'UTRs pourrait réguler la fonction des protéines en augmentant ainsi la complexité du vivantOver time, life has evolved to produce remarkably complex organisms. To cope with this complexity, organisms have evolved a plethora of regulatory mechanisms. For instance, thousands of long non-coding RNAs (lncRNAs) are transcribed by mammalian genomes, presumably expanding their regulatory capacity. An emerging concept is that lncRNAs can serve as protein scaffolds, bringing proteins in proximity, but the prevalence of this mechanism is yet to be demonstrated. In addition, for every messenger RNA encoding a protein, regulatory 3’ untranslated regions (3’UTRs) are also present. Recently, 3’UTRs were shown to form protein complexes during translation, affecting the function of the protein under synthesis. However, the extent and importance of these 3’UTR-protein complexes in cells remains to be assessed.This thesis aims to systematically discover and provide insights into two ill-known regulatory mechanisms involving the non-coding portion of the human transcriptome. Concretely, the assembly of protein complexes promoted by lncRNAs and 3’UTRs is investigated using large-scale datasets of protein-protein and protein-RNA interactions. This enabled to (i) predict hundreds of lncRNAs as possible scaffolding molecules for more than half of the known protein complexes, as well as (ii) infer more than a thousand distinct 3’UTR-protein complexes, including cases likely to post-translationally regulate moonlighting proteins, proteins that perform multiple unrelated functions. These results indicate that a high proportion of lncRNAs and 3’UTRs may be employed in regulating protein function, potentially playing a role both as regulators and as components of complexity
11
Titus, Mitchell. „Subunit interactions and protein-DNA interactions of the Drosophila melanogaster small nuclear RNA activating protein complex“. Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p3283558.
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Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2007.Title from first page of PDF file (viewed Nov. 21, 2007). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.
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Takeuchi, Akiko Krol Alain Allmang-Cura Christine. „RNA-protein interaction in the selenoprotein synthesis machinery“. Strasbourg : Université de Strasbourg, 2009. http://eprints-scd-ulp.u-strasbg.fr:8080/1133/01/TAKEUCHI_Akiko_2009.pdf.
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Thèse de doctorat : Sciences du Vivant. Aspects moléculaire et cellulaire de la Biologie : Strasbourg : 2009.Thèse soutenue sur un ensemble de travaux. Titre provenant de l'écran-titre. Bibliogr. 11 p.
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Méthot, Nathalie. „RNA and protein interactions by eIF4B during translation initiation“. Thesis, McGill University, 1996. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=40401.
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One of the most enduring questions pertaining to eukaryotic translation initiation is how the 40S ribosomal subunit recognizes and binds at or near the cap-structure of mRNAs. Eukaryotic initiation factor 4B (eIF4B) is one of the factors that are required for this step of protein synthesis. eIF4B stimulates the RNA helicase activity of eIF4A and eIF4F, melting RNA secondary structure in the 5$ sp prime$ untranslated region (UTR), and is thus believed to contribute to ribosome binding by creating an area of single-stranded RNA accessible to the 40S ribosomal subunit We studied the mechanism of action of eIF4B by initiating a structure-function analysis of this factor. An RNA binding site, located in the carboxy-terminal end between amino acids 367 and 423, was found essential for non-specific RNA binding and eIF4A helicase stimulation. This region is distinct and independent from the canonical RNA Recognition Motif (RRM) located near the amino-terminus. The latter plays no role in non-specific RNA binding and has little impact on the eIF4A helicase stimulation. A self-association region located between residues 213-312 was identified. This segment is rich in aspartic acid, arginine, tyrosine and glycine (DRYG) residues, and can self-associate independently from other regions of eIF4B. The DRYG domain also interacts directly with the p170 subunit of eIF3. Finally, iterative in vivo RNA selection demonstrated that the eIF4B RRM is functional and binds specifically to RNA stem-loop structures. The RRM also associates with 18S rRNA. eIF4B possesses two independent RNA binding sites and associates with two different RNA molecules simultaneously. We conclude that eIF4B is organized into three distinct domains: the carboxy-terminal eIF4A RNA helicase stimulatory domain, the DRYG dimerization and eIF3 p170 interaction domain, and the RRM. Furthermore, two additional mechanisms by which eIF4B could stimulate ribosome binding to the mRNA are now apparent. eIF4B may target t
14
Bachand, François. „Functional reconstitution and RNA-protein interactions of human telomerase“. Thesis, McGill University, 2002. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38460.
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Telomerase is a ribonucleoprotein (RNP) enzyme responsible for the replenishment of repetitive DNA sequences present at the ends of most eukaryotic chromosomes. Telomerase is minimally composed of a protein catalytic subunit, the telomerase reverse transcriptase (TERT), and an RNA subunit. Using a small single-stranded segment (7--11 nucleotides) of the telomerase RNA as a template, the active site of the TERT catalytic subunit adds complementary nucleotides onto telomeric DNA. The primary goal of the work presented in this thesis was to biochemically and functionally characterize the human telomerase reverse transcriptase (hTERT) and the human telomerase RNA (hTR), as well as to identify novel telomerase-associated proteins. First, we demonstrated that the budding yeast Saccharomyces cerevisiae possesses the cellular machinery to fully assemble a catalytically active human telomerase RNP in vivo. We also analyzed telomerase activity and binding of hTR to hTERT in rabbit reticulocyte lysates by expressing different hTERT and hTR variants. Our results identified two distinct regions of hTR that can independently bind hTERT in vitro. Furthermore, sequences or structures that include the conserved CR4--CR5 domain of hTR were found to be important for hTERT-hTR interactions and telomerase activity reconstitution. Human TERT carboxy- and amino-terminal amino acid deletions indicated that the polymerase and RNA binding functions of hTERT are separable. We also found that the product of the survival of motor neuron ( SMN) gene, a protein involved in the biogenesis of certain RNPs, is a telomerase-associated protein. Our results demonstrate that the human TR and the human TERT are not associated with Sm proteins, in contrast to Saccharomyces cerevisiae telomerase. Taken together, the work presented in this thesis indicate that the reconstitution of human telomerase activity in vitro requires regions of hTERT that (i) are distinct from the conserved reverse transcriptase
15
Sohrabi-Jahromi, Salma [Verfasser]. „Quantitative Modeling of RNA-Protein Interactions / Salma Sohrabi-Jahromi“. Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1233481533/34.
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16
Haussmann, Irmgard Ursula. „RNA-protein interactions in the U7 small nuclear Ribonucleoprotein /“. [S.l.] : [s.n.], 1994. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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17
Friedel, Caroline Christina. „Analysis of High-Throughput Data - Protein-Protein Interactions, Protein Complexes and RNA Half-life“. Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-96883.
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18
Takeuchi, Akiko. „RNA-protein interaction in the selenoprotein synthesis machinery“. Strasbourg, 2009. http://www.theses.fr/2009STRA6054.
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La sélénocystéine est incorporée co-traductionnellement dans les sélénoprotéines en réponse à un codon UGA habituellement l’un des 3 codons stop. La protéine SBP2 joue un rôle majeur dans ce mécanisme de recodage en se liant à une structure en tige-boucle (SECIS) située dans la région 3’UTR de l’ARNm des sélénoprotéines. Nous avons isolé et caractérisé fonctionnellement SBP2 de Drosophila melanogaster. Par comparison avec SBP2 humaine, nous avons identifié un domaine de liaison à l’ARN additionnel essentiel à la liaison au SECIS et à la sous-unité ribosomique 60S et permettant une sélectivité structurale du SECIS. Des prédictions structurales et des analyses biophysiques ont établi que SBP2 était une protéine globalement désordonnée ou “Intrinsically Disordered Protein” qui ne se replie qu’en présence de partenaires. Enfin, nous avons établi que l’assemblage des mRNP de sélénoprotéines faisait appel à des facteurs communs et présentait de multiples similarités avec celui des sn/snoRNPThe 21st amino acid selenocysteine is encoded by a UGA codon that usually signifies translational termination. Selenoprotein synthesis therefore requires specialized factors. Among these is SBP2 that binds the SECIS, a stem-loop structure in the 3’UTR of selenoprotein mRNAs. In structural analyses of SBP2, we isolated and functionally characterized Drosophila melanogaster SBP2. By comparing it with human SBP2, we identified an additional RNA binding domain that is essential for SECIS and 60S ribosomal subunit binding, and also enables SECIS structure selectivity. In addition, computational and biophysical analyses established that SBP2 is globally unfolded, supporting our hypothesis that SBP2 is an Intrinsically Disordered Protein and becomes folded in the presence of partners yet to be identified. Finally, we searched for potential partners of SBP2 and our results showed that the molecular assembly of selenoprotein mRNPs has many similarities with that of sn/snoRNPs
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Kim, Young-Chan. „Protein-ligand and protein-protein interactions involved in de novo initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp)“. [Bloomington, Ind.] : Indiana University, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&res_dat=xri:pqdiss&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&rft_dat=xri:pqdiss:3204540.
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Thesis (Ph. D.)--Indiana University, Dept. of Chemistry, 2006.Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0249. Adviser: C. Cheng Kao. "Title from dissertation home page (viewed Feb. 9, 2007)."
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Cass, Danielle Marie. „Role of two RNA binding properties in pre-mRNA splicing /“. view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950811&sid=2&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 67-80). Also available for download via the World Wide Web; free to University of Oregon users.
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Huang, Ching-jung. „The role of HnRNP proteins, PSF and nonO/p54[superscript nrb], in pre-mRNA binding and splicing /“. Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004292.
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22
Chan, Yin-tung Crystal, und 陳燕彤. „Demonstration of specific physical interaction between CHOP mRNA and intracellular proteins“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2011. http://hub.hku.hk/bib/B47169369.
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The ability of a cell to respond precisely to environmental stress depends on the expression of a large number of genes in a finely coordinated manner. One of such genes is CHOP that encodes the CCAAT/Enhancer-Binding Protein Homologous Protein. CHOP is usually expressed to mediate apoptosis under the condition of excessive stress. The expression of CHOP therefore has to be stringently regulated as its expression will determine the fate of a cell under stress. The expression of many genes is regulated at the posttranscriptional level through the metabolism of their mRNA, such as maturation, transport, storage, and degradation of mRNA. Many metabolic processes of mRNA are known to be mediated by RNA-binding proteins that specifically interact with the mRNA. RNA-binding proteins that interact with the CHOP mRNA have until present not been identified. The aim of this study is to investigate what proteins may bind specifically to CHOP mRNA. The study will enable further understanding regarding how the expression of CHOP is regulated in cellular stress response.
Proteins extracted from HeLa cells were incubated with a 335bp [3H]-labelled CHOP RNA probe that spans over a part of the coding region and the 3’UTR of CHOP mRNA. Sucrose density gradient ultracentrifugation revealed that after incubation with proteins extracted from HeLa cells, the sedimentation rate of the [3H]-CHOP RNA probe was significantly higher than that of the free [3H]-RNA probe. The formation of heavy molecular complexes involving the [3H]-CHOP RNA probe was therefore suggested. However, no increase in sedimentation rate of the [3H]-CHOP RNA probe was observed in the presence of an excess of unlabelled CHOP RNA probe. Similar observations were made when the experiments were performed using proteins isolated from cells treated with As2O3.
Two putative sequence elements, the Adenylate-Uridylate-Rich Element (ARE) and the Putative Regulatory Element (PRE) located respectively in the 3’UTR and coding region of the CHOP mRNA were then examined for their involvement in RNA-protein interaction. The deletion of ARE and/or PRE, from the [3H]-CHOP RNA probe had little effect on the binding of the RNA probe to the HeLa cell proteins. Consistently, unlabelled CHOP RNA probes with the same deletions were only slightly weaker in competing with the intact [3H]-CHOP RNA probe to bind to HeLa cell proteins. Human Antigen R (HuR) was identified by Western blot analysis to be present in the proteins that were obtained by pull-down assays using biotinylated CHOP RNA as a probe. The deletion of ARE and/or PRE resulted in a slight reduction of HuR obtained by pull down assays.
This study provides the first evidence that physical binding interaction occurs between intracellular RNA-binding proteins and CHOP mRNA. More importantly, one such protein is HuR. Data suggest that HuR binding to the CHOP mRNA is mediated by sequences in the CHOP mRNA other than ARE and PRE.published_or_final_version
Biochemistry
Master
Master of Philosophy
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Lymperopoulos, Konstantinos. „Functional characterisation of the bluetongue virus non-structural protein NS2-protein and RNA-protein interactions“. Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424733.
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Bennett, N. „The role of p2 in protein-RNA interactions of HIV“. Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596569.
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The two subtypes of the Human Immunodeficiency Virus, HIV-1 and HIV-2, differ in the way that they package their genomic RNA with the structural Gag protein. HIV-1 packages its genomic RNA predominantly in a trans manner, and can cross-package HIV-2 RNA in co-transfections. Conversely, HIV-2 is not capable of packaging HIV-1 RNA reciprocally, and packages its own RNA using a co-translational mechanism. Chimeric viruses where the RNA-binding domain of HIV-1 is substituted into the structural of Gag protein of HIV-2 appear to transfer the trans packaging phenotype, but this function is only maximally present when the adjacent p2 peptide is also included. The aim of the work presented here was to investigate possible direct and indirect effects of the p2 domain in RNA-protein capture in HIV-1 and HIV-2. Chimeric Gag proteins were expressed using bacterial systems and their interaction with in vitro transcribed RNA of the HIV leader sequences investigated using GST-Pulldown assays, UV-crosslinking and mobility shift assays. Evidence is presented that is consistent with a complex and possibly cooperative binding interaction occurring between Gag and the packaged RNA, which correlates to some extent with the presence of the p2 domain. The subcellular location of the Gag protein was investigated using transfection with Gag-expressing and viral constructs, following by confocal microscopy of the immunostained cells, and no difference was found that could be attributed to the p2 domain. Additionally, the replication characteristics of the chimeric viruses were examined in short and long term culture. All chimeric viruses were defective in short term culture, but one culture did produce a replication-competent strain on long-term passage. This strain had wild type replication kinetics, and sequencing of the revertant virus revealed a single-base substitution in the highly basic region of the MA domain of Gag.
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Talbot, Simon John. „Structural studies of RNA-protein interactions in the bacteriophage MS2“. Thesis, University of Leeds, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.303328.
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Gale, Andrew J. (Andrew John). „Protein-RNA domain-domain interactions in a tRNA sythetase system“. Thesis, Massachusetts Institute of Technology, 1995. http://hdl.handle.net/1721.1/39369.
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Jung, Jeenah. „Development of optical imaging method for detecting RNA-protein interactions“. Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/54278.
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The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.
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Chen, Cai. „Quantitative studies of RNA editing and nucleosomal DNA-protein interactions“. The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1417523347.
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Wu, Jiang. „Functional studies of mouse quaking protein /“. Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3008472.
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30
Lee, Semin. „Molecular characterization of protein-nucleic acid interfaces : applications in bioinformatics“. Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609284.
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Henscheid, Kristy L. „Functional conservation and RNA binding of the pre-mRNA splicing factor U2AF65 /“. view abstract or download file of text, 2007. http://proquest.umi.com/pqdweb?did=1400950821&sid=5&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 129-141). Also available for download via the World Wide Web; free to University of Oregon users.
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Defenbaugh, Dawn. „Analysis of hepatitis delta virus RNA structure effects on RNA-protein interactions and viral replication /“. Connect to Electronic Thesis (CONTENTdm), 2008. http://worldcat.org/oclc/459757001/viewonline.
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Murray, Jill Isobel. „Identification of motifs that function in the splicing of non-canonical introns /“. Connect to title online (ProQuest), 2007. http://proquest.umi.com/pqdweb?did=1453227351&sid=1&Fmt=2&clientId=11238&RQT=309&VName=PQD.
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Thesis (Ph. D.)--University of Oregon, 2007.Typescript. Includes vita and abstract. Includes bibliographical references (leaves 76-84). Also available online in ProQuest, free to University of Oregon users.
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Rajendran, Kottampatty. „DISSECTING THE FUNCTIONS OF CARMOVIRUS AND TOMBUSVIRUS REPLICASE PROTEINS“. UKnowledge, 2004. http://uknowledge.uky.edu/gradschool_diss/432.
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Replication of genetic material is the most important and central process during the viral life cycle. Most RNA viruses assign one or more proteins translated from their own genome for replicating genomic RNAs. Understanding the various biochemical activities of these replication proteins is the aim of this dissertation research. The replicase proteins of Turnip crinkle virus (TCV) and Tomato bushy stunt virus (TBSV) were selected for this study. Both viruses have small, messenger-sense, single-stranded RNA genomes. Replicase proteins p28/p88 of TCV and p33/p92 of TBSV- were expressed and purified from E. coli as N-terminal fusions to maltose binding protein. In vitro assays revealed that the recombinant p88 has RNA-dependent RNA polymerase (RdRp) and RNAbinding activities. Deletion of the N-terminal p28 domain in p88 resulted in a highly active RdRp, while further deletions at both N- and C-terminal ends abolished RdRp activity. Comparison of p88, the N-terminal p28-deletion mutant of p88 and a TCV RdRp preparation obtained from infected plants revealed remarkable similarities in RNA template recognition and plus and minus strands synthesis. Contrary to recombinant TCV RdRp activities under similar experimental conditions. p33 preferentially binds to singlestranded (ss) RNA with positive cooperativity in vitro. The RNA binding activity was mapped to arginine/proline-rich motif (RPR-motif) at the C-terminus of p33 and the corresponding sequence in p92. The non-overlapping C-terminal domain of p92 also contained additional RNA-binding regions that flank the conserved RdRp motifs on both sides. Cooperative RNA binding by p33 suggested inter-molecular interactions between p33 monomers. Indeed the yeast two-hybrid and surface plasmon resonance assays revealed interactions between p33 and p33 and also between p33 and p92. The sequence involved in the protein-protein interactions was mapped to the C-terminal region in p33, proximal to RPR-motif. Within this region, mutations introduced at two short stretches of amino acid residues were found to affect p33:p33 and p33:p92 interactions in vivo and also decreased the replication of a TBSV-defective interfering RNA in yeast, a model system, supporting the significance of these protein interactions in tombusvirus replication.
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Ramesh, Arati. „Structural studies of the Ro ribonucleoprotein and the metalloregulator CsoR“. Thesis, [College Station, Tex. : Texas A&M University, 2007. http://hdl.handle.net/1969.1/ETD-TAMU-1426.
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Fan, Yan Baranger Anne M. Katzenellenbogen John A. Zhao Huimin Silverman Scott K. „Exploring protein-RNA interactions with site-directed mutagenesis and phage display“. Urbana, IL.: University of Illinois, 2009. http://hdl.handle.net/2142/14755.
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Militti, Cristina 1982. „Drosophila UNR regulates dosage compensation through modulation of RNA-protein interactions“. Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/283476.
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En Drosophila, el desequilibrio en cuanto al contenido de genes ligados al cromosoma X entre hembras (XX) y machos (XY) es corregido mediante la duplicación de la transcripción del único cromosoma X del macho. Este proceso, llamado compensación de dosis, es mediado por un ensamblaje molecular compuesto por al menos cinco proteínas (MSL1, MSL2, MSL3, MLE y MOF) y dos RNAs largos no codificantes (roX1 y roX2), llamado complejo de compensación de dosis (DCC). La compensación de dosis requiere dos condiciones fundamentales: el reconocimiento específico del cromosoma X por el DCC, y la restricción del proceso a moscas macho.
La proteína de unión a RNA Upstream-of-N-Ras (UNR) está implicada en la consecución de ambas condiciones, y aquí hemos estudiado los mecanismos moleculares por los que UNR actúa. Hemos encontrado que, en machos, UNR promueve la compensación de dosis facilitando la asociación de roX2 a MLE, necesaria para una correcta formación del DCC y para su unión al cromosoma X. En hembras, UNR inhibe la compensación de dosis, al menos en parte, promoviendo la unión de SXL al extremo 3’ UTR del mRNA que codifica para msl2, lo que resulta en represión de la traducción de msl2 e inhibición de la formación del DCC.In Drosophila, the imbalance in X-linked gene content between females (XX) and males (XY) is restored through the 2-fold hypertranscription of the single male X-chromosome. This process, which is called dosage compensation, is mediated by the action of the dosage compensation complex (DCC), a ribonucleoprotein assembly composed of at least five proteins (MSL1, MSL2, MSL3, MLE and MOF) and two long non-coding RNAs (roX1 and roX2). Two features are essential for correct dosage compensation: the specific recognition of the X-chromosome by the DCC and the confinement of the DCC function to the male organism. The RNA binding protein Upstream of N-ras (UNR) is involved in the regulation of these two processes and we have dissected the molecular mechanisms by which this regulation occurs. We have found that, in male flies, UNR promotes dosage compensation by facilitating the association of roX2 with MLE, which is required for correct DCC formation and X-chromosome targeting. In female flies, UNR represses dosage compensation in part by enhancing the binding of SXL to the 3’UTR of msl2 mRNA, thus ensuring tight msl2 translational repression and subsequent inhibition of DCC formation.
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Armaos, Alexandros 1989. „Computational characterization of protein-RNA interactions and implications for phase separation“. Doctoral thesis, Universitat Pompeu Fabra, 2020. http://hdl.handle.net/10803/668546.
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Despite what was previously considered, the role of RNA is not only to carry the genetic
information from DNA to proteins. Indeed, RNA has proven to be implicated in more
complex cellular processes. Recent evidence suggests that transcripts have a regulatory
role on gene expression and contribute to the spatial and temporal organization of the
intracellular environment. They do so by interacting with RNA-binding proteins (RBPs)
to form complex ribonucleoprotein (RNP) networks, however the key determinants that
govern the formation of these complexes are still not well understood. In this work, I will
describe algorithms that I developed to estimate the ability of RNAs to interact with
proteins. Additionally, I will illustrate applications of computational methods to propose
an alternative model for the function of Xist lncRNA and its protein network.
Finally, I will show how computational predictions can be integrated with high
throughput approaches to elucidate the relationship between the structure of the RNA and
its ability to interact with proteins. I conclude by discussing open questions and future
opportunities for computational analysis of cell’s regulatory network.
Overall, the underlying goal of my work is to provide biologists with new insights into
the functional association between RNAs and proteins as well as with sophisticated tools
that will facilitate their investigation on the formation of RNP complexesA pesar de lo que se consideraba anteriormente, el papel del ARN no es solo transportar la información genética del ADN a las proteínas. De hecho, el ARN ha demostrado estar implicado en muchos procesos celulares más complejos. La evidencia reciente sugiere que los transcriptos tienen un papel regulador en la expresión génica y contribuyen a la organización espacial y temporal del entorno intracelular. Lo hacen interactuando con proteínas de unión a ARN (RBP) para formar redes complejas de ribonucleoproteína (RNP), sin embargo, los determinantes clave que rigen la formación de estos complejos aún no se conocen bien. En este trabajo, describiré algoritmos que he desarrollado para estimar la capacidad de los ARN de interactuar con las proteínas. Además, ilustraré aplicaciones de métodos computacionales para proponer una maquinaria alternativa para el Xist lncRNA y su red de interacciones. Finalmente, mostraré cómo las predicciones computacionales pueden integrarse con enfoques de alto rendimiento para dilucidar la relación entre la estructura del ARN y su capacidad para interactuar con las proteínas. Concluyo discutiendo preguntas abiertas y oportunidades futuras para el análisis computacional de la red reguladora de la célula. En general, el objetivo subyacente de mi trabajo es proporcionar a los biólogos nuevas ideas sobre la asociación funcional entre ARN y proteínas, así como herramientas sofisticadas que facilitarán su investigación sobre la formación de complejos RNP.
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Urena, Gonzalez Luis. „Identification of RNA-protein interactions involved in the norovirus life cycle“. Thesis, Imperial College London, 2012. http://hdl.handle.net/10044/1/39844.
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Members of the Caliciviridae family, including murine norovirus (MNV), contain conserved RNA secondary structure elements located at the 5' and 3' regions of the viral genome. The 5' extremity of the genomic (5'G) and subgenomic (5'SG) RNAs, as well as the 3' extremity (3'Ex), are believed to be involved in many aspects of calicivirus life cycle. This project was designed to identify proteins interacting with the extremities of MNV genome, as this would include proteins involved in viral translation and replication. We used a riboproteomics method to isolate RNA-binding proteins by RNA affinity chromatography and mass spectrometry from RAW264.7 S-100 extracts and rabbit reticulocyte lysates (RRL). Combining data sets obtained from RRL and S-100 lysates; various unique cellular host factors were identified using the 5'G, 5'SG, and 3'Ex RNA targets. These included several RNA-binding proteins involved in other positive-strand virus replication but not reported for Caliciviridae family, including Y-box binding protein 1 (YBX-1), ATP- dependent RNA helicase (DDX3), and Heat Shock Protein 90 (HSP90). Other identified proteins isolated as binding to the MNV genome included known calicivirus interacting proteins such as La protein (La), poly(rC) binding protein 1 (PCBP) and polypyrimidine tract binding protein (PTB). The colocalisation of some of the proteins was confirmed by confocal microscopy. The functional role of a subset of identified proteins was further analysed by using RNAi and specific protein inhibitors. These results indicate that knockdown of La, PTB, YBX-1, DDX3 and HSP90 dramatically reduces viral titre and RNA replication. Confocal microscopy showed that some of these host factors redistribute to the site of infection colocalising with MNV replication components. The use of DDX3 and HSP90 inhibitors demonstrated a significant effect on infectious virus production confirming that these factors play a crucial role in the virus life cycle. This work highlights how screening of RNA-binding proteins in the context of MNV by a riboproteomics approach could well provide insights in the mechanisms for the regulation of viral translation and replication, and could potentially pinpoint new drug targets for these important pathogens.
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Osborne, Jane C. „Interactions of Bunyamwera virus nucleocapsid protein and encapsidation of viral RNA“. Thesis, University of Glasgow, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.341712.
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Karakasiliotis, Ioannis. „Analysis of RNA-protein interactions involved in calicivirus translation and replication“. Thesis, Imperial College London, 2008. http://hdl.handle.net/10044/1/1304.
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The interaction of host-cell nucleic acid-binding proteins with the genomes of positive-stranded RNA viruses is known to play a role in the translation and replication of many viruses. To date, however, the characterisation of similar interactions with the genomes of members of the Caliciviridae family has been limited to in vitro binding analysis. In this study, feline calicivirus (FCV) and murine norovirus (MNV) have been used as model systems to identify and characterise the role of host-cell factors that interact with the viral RNA and RNA structures that regulate virus replication. It was demonstrated that RNA-binding proteins such as polypyrimidine tract-binding protein (PTB), poly(C)-binding proteins (PCBPs) and La protein interact with the extremities of MNV and FCV genomic and subgenomic RNAs. PTB acted as a negative-regulator in FCV translation and is possibly involved in the switch between translation and replication during the late stages of the infection, as PTB is exported from the nucleus to the cytoplasm, where calicivirus replication takes place. Furthermore, using the MNV reverse-genetics system, disruption of 5' end stem-loops reduced infectivity ~15-20 fold, while disruption of an RNA structure that is suspected to be part of the subgenomic RNA synthesis promoter and an RNA structure at the 3' end completely inhibited virus replication. Restoration of infectivity by repair mutations in the subgenomic promoter region and the recovery of viruses that contained repressor mutations within the disrupted structures, in both the subgenomic promoter region and the 3' end, confirmed a functional role for these RNA secondary structures. Overall this study has yielded new insights into the role of RNA structures and RNA-protein interactions in the calicivirus life cycle.
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Bailey, Daniel John. „Cellular proteins in picornavirus replication“. Thesis, University of Reading, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298484.
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Loushin, Newman Carrie Lee. „Characterization of QKI RNA binding function /“. Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004323.
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Lee, Jae-Hyung. „Analysis of protein-RNA and protein-peptide interactions in Equine Infectious Anemia Virus (EIAV) infection“. [Ames, Iowa : Iowa State University], 2007.
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Deutsch, Christopher Wayne. „Discovery and Characterization of the Proteins Involved in the Synthesis of N⁶-Threonylcarbamoyl Adenosine, a Nucleoside Modification of tRNA“. PDXScholar, 2016. http://pdxscholar.library.pdx.edu/open_access_etds/3080.
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N6-threonylcarbamoyl adenosine (t6A) is a universally conserved tRNA modification found at position 37 of tRNAs which decode ANN codons. Structural studies have implicated its presence as a requirement for the disruption of a U-turn motif in certain tRNAs, leading to the formation of properly structured anticodon stem loop. This structure is proposed to enhance the base pairing between U36 of tRNA and A1 of the codon which aids in translational frame maintenance.
Despite significant effort since its discovery in the 1970s the enzymes involved in its biosynthesis remained undiscovered. Bioinformatic analysis identified two proteins as likely candidates for t6A synthesis, YrdC and YgjD. Subsequent gene knockout experiments in yeast were consistent with their involvement in t6A biosynthesis in vivo. Furthermore, clustering between the bacterial genes ygjD, yeaZ and yjeE as well as the identification of a protein interaction network between YgjD, YeaZ, and YjeE suggested that YeaZ and YjeE might be involved in t6A biosynthesis.
The genes encoding ygjD, yeaZ, yrdC and yjeE were cloned from E. coli and the recombinant protein was purified. Experiments analyzing the incorporation of [U-14C]-L-threonine and [14C]-bicarbonate (substrates previously indicated in its biosynthesis) into tRNA in the presence of these four proteins demonstrated the first reconstitution of the t6A pathway in vitro. LC-MS analysis verified the formation of t6A, and these proteins were renamed TsaD (YgjD), TsaB (YeaZ), TsaC (YrdC), and TsaE (YjeE).
Biochemical characterization of this pathway suggested that the formation of t6A proceeds through an unstable threonylcarbamoyl adenosine monophosphate (TC-AMP) intermediate, which is produced by TsaC from its substrates CO2, L-threonine and ATP. To investigate this reaction in more detail a coupled assay was developed, enabling sensitive detection of turn over. TsaC is a promiscuous enzyme which readily accepts several amino acids as substrates. The formation of t6A from TC-AMP is catalyzed by TsaD, TsaB, and TsaE. Of these three proteins only TsaD is universally conserved suggesting it is the protein catalyzing the transfer of the threonylcarbamoyl moiety to A37 of tRNA. This transfer is not promiscuous as only TC-AMP serves as an efficient substrate for t6A formation. Structural investigation of these proteins are consistent with the formation of a single protein complex potentially alleviating issues with the reactivity and instability of TC-AMP.
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Rajendran, KS. „Dissecting the functions of carmovirus replicase proteins dissecting the functions of carmovirus tombusvirus replicase proteins dissecting“. Lexington, Ky. : [University of Kentucky Libraries], 2004. http://lib.uky.edu/ETD/ukyplpa2004d00150/Rajendra.pdf.
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Thesis (Ph. D.)--University of Kentucky,2004.Title from document title page (viewed Oct. 12, 2004). Document formatted into pages; contains ix, 111 p. : ill. Includes abstract and vita. Includes bibliographical references (p. 97-110).
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Tsai, Hsin-Yue. „Elucidating the role of protein cofactors in RNA catalysis using ribonuclease P as the model system“. Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1141769728.
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Sidiqi, Mahjooba. „The structure and RNA-binding of poly (C) protein 1“. University of Western Australia. School of Biomedical, Biomolecular and Chemical Sciences, 2008. http://theses.library.uwa.edu.au/adt-WU2008.0077.
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[Truncated abstract] Regulation of mRNA stability is an important posttranscriptional mechanism involved in the control of gene expression. The rate of mRNA decay can differ greatly from one mRNA to another and may be regulated by RNA-protein interactions. A key determinant of mRNA decay are sequence instability (cis) elements often located in the 3' untranslated region (UTR) of many mRNAs. For example, the AU rich elements (AREs), are such well characterized elements, and most commonly involved in promoting mRNA degradation, and specific binding of proteins to these elements leading to the stabilization of some mRNAs. Other cis-elements have been described for mRNA in which mRNA stability is a critical component of gene regulation. This includes the androgen receptor (AR) UC-rich cis element in its 3'UTR. The AR is a key target for therapeutics in human prostate cancer and thus understanding the mechanism involved in regulating its expression is an important goal. The [alpha]CP1 protein, a KH-domain containing RNA-binding protein has been found to bind this UC-rich region of the AR and is thought to play an important role in regulating AR mRNA expression. [alpha]CP1 protein is a triple KH (hnRNP K homology) domain protein with specificity for Crich tracts of RNA and ssDNA (single stranded DNA). Relatively little is known about the structural interaction of [alpha]CP1 with target RNA cis elements, thus the present study aimed to better understand the nature of interaction between 30 nt 3'UTR UC-rich AR mRNA and [alpha]CP1 protein using various biophysical techniques, in an attempt to determine which [alpha]CP1 domain or combination of domains is involved in RNA-binding. These studies could ultimately provide novel targets for drugs aimed to regulate AR mRNA expression in prostate cancer cells. At the commencement of this study little was known about the structure of the [alpha]CP1- KH domains and their basis for poly (C) binding specificity. ... Additional studies addressed the significance of the four core recognition nucleotides (TCCC) using a series of cytosine to thymine mutants. The findings verified some of the results predicted from structural studies, especially the need for maximum KH binding to a core tetranucleotide recognition sequence. Our mutational studies of the four core bases confirmed the importance of cytosine in positions two and three as no binding was observed, while some binding was observed when the fourth base was mutated. In summary, the work presented in this thesis provides new detailed insight into the molecular interactions between the [alpha]CP1-KH domain and AR mRNA. Furthermore, these studies shed light on the nature of protein/mRNA interactions in general, as well as the specific complex that forms on AR mRNA. These studies have provided new understanding into the mode of [alpha]CP1 binding at a target oligonucleotide binding site and, provide a foundation for future studies to define structure of multiprotein/oligonucleotide complexes involved in AR mRNA gene regulation. Understanding the detailed interaction between the AR mRNA and [alpha]CP1 could provide possible targets for drug development at reducing AR expression in prostate cancer cells by interfering with the interaction of [alpha]CP1 and AR-mRNA.
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Moran-Jones, Kim. „hnRNPs A2 and A3 : nucleic acid interactions /“. St. Lucia, Qld, 2004. http://www.library.uq.edu.au/pdfserve.php?image=thesisabs/absthe17983.pdf.
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Dry, Inga Ruth. „Functional analysis of viral RNA and protein-RNA interactions involved in the replication of poliovirus type 3“. Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.409998.
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