Dissertations / Theses: 'RNA polymerases'

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Salgado, Maria Paula Santos Cordeiro. "Structural studies of RNA-dependent RNA polymerases." Thesis, Open University, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.430559.

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Forrest, David Andrew. "Novel types of RNA polymerases." Thesis, University of Newcastle upon Tyne, 2015. http://hdl.handle.net/10443/2808.

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Transcription, the first step of gene expression, is accomplished in all domain of life by the multisubunit RNA polymerase (RNAP). Accordingly, the RNAP is an ancient enzyme that is highly conserved in all cellular organisms. However, in-depth bioinformatics has led to the identification of proteins distantly related to the multisubunit RNAP, such as YonO and ORF6 RNAP. Whilst being putative single subunit RNAPs, YonO and ORF6 RNAP are completely unrelated to T7 RNAP. YonO, encoded by the Bacillus subtilis SPβ prophage, has incredibly little similarity to RNAP. Conversely, ORF6 RNAP belonging to the Kluyveromyces lactis killer system contains half of the conserved RNAP active centre. YonO and ORF6 RNAP are potentially novel RNAPs and their characterisation will give new insights into mechanisms of transcription as well as the biology of the organisms which they belong to. Despite lacking most of the conserved RNAP active centre, we have shown YonO is a very efficient DNA dependant RNAP. Striking, unlike all of its multisubunit relatives, YonO is able to initiate transcription on double stranded DNA without accessory factors (such as σ factors in bacteria). Furthermore, our work suggests YonO is expressed during induction of SPβ and functions to transcribe the SPβ late genes. This potentially makes YonO the first bacteriophage single subunit RNAP that resembles the multisubunit RNAP. On the other hand, ORF6 RNAP was very poor at polymerisation, with or without its putative accessory subunit. This suggests additional, currently unknown proteins are required for RNAP activity and potentially a new molecular mechanism of RNA polymerisation. YonO homologues exist in a wide range of bacteria including fermicutes and cyanobacteria. YonO represents a new class of RNAP and our discoveries potentially open up a new area of research as well as being potentially useful for biotechnology and synthetic biology. In contrast, transcription by ORF6 RNAP is more complex than previously thought, with alternative lines of investigation required to identify additional factors that contribute to ORF6 RNAP activity.

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Wright, Sam Mathew. "Structural and biophysical studies of RNA-dependent RNA polymerases." Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:d5c2a16d-e1e2-4c22-aca5-70f72aa96853.

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RNA-dependent RNA polymerases (RdRps) play a vital role in the life cycle of RNA viruses, being responsible for genome replication and mRNA transcription. In this thesis viral RdRps (vRdRps) of dsRNA bacteriophage phi6 (phi6 RdRp) and Severe Acute Respiratory Syndrome (SARS) coronavirus [non structural protein 12 (NSP-12)] are studied. For SARS polymerase NSP-12, a library-based screening method known as ESPRIT (Expression of Soluble Protein by Random Incremental Truncation) was employed in an attempt to isolate domains of NSP-12 that express solubly in Escherichia coli (E. coli) and are thereby suitable for structural studies. This experiment identified for the first time in a systematic fashion, conditions under which the SARS polymerase could be solubly expressed at small scale and allowed mapping of domain boundaries. Further experiments explored different approaches for increasing expression levels of tractable fragments at large scale. Bacteriophage phi6 RdRp is one of the best studied vRdRps. It initiates RNA synthesis using a de novo mechanism without the need for a primer. Although formation of the de novo initiation complex has been well studied, little is known about the mechanism for the transition from initiation to elongation (i.e. extension of an initiated dinucleotide daughter strand). In the phi6 RdRp initiation complex the C-terminal domain (CTD) blocks the exit path of the newly synthesised dsRNA which must be displaced for the addition of the third nucleotide. The crystal structure of a C-terminally truncated phi6 RdRp (P2T1) reveals the strong non-covalent interactions between the CTD and the main body of the polymerase that must be overcome for the elongation reaction to proceed. Comparing new crystal structures of complexes of both wild-type (WT) and a mutant RdRp (E634 to Q, which removes a salt-bridge between the CTD and main body of the polymerase) with various oligonucleotides (linear and hairpin), nucleoside triphosphates (NTPs) and divalent cations, alongside their biophysical and biochemical properties, provides an insight into the precise molecular details of the transition reaction. Thermal denaturation experiments reveal that Mn2+ acquired from the cell and bound at the phi6 RdRp non-catalytic ion site sufficiently weakens the polymerase structure to facilitate the displacement of the CTD. Our crystallographic and biochemical data also indicate that Mn2+ is released during this displacement and must be replaced for the elongation to proceed. Our data explain the role of the non-catalytic divalent cation in vRdRps and pinpoint the Mn2+-dependent step in viral replication. In addition, by inserting a dysfunctional Mg2+ at the non-catalytic ion site for both WT and E634Q RdRps we captured structures with two NTPs bound within the active site in the absence of Watson-Crick base pairing with template and could map movements of divalent cations during preinitiation through to initiation. Oligonucleotides present on the surface of phi6 RdRp allowed mapping of key residues involved in template entry and unwinding of dsRNA; these preinitiation stages have not been observed previously. Considering the high structural homology of phi6 RdRp with other vRdRps, particularly from (+)ssRNA hepatitis C virus (HCV), insights into the mechanistic and structural details of phi6 RdRp are thought to be relevant to the general understanding of vRdRps.

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Chan, Annie Yee-Man. "Interactions between the influenza virus RNA polymerase and cellular RNA polymerase II." Thesis, University of Oxford, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670083.

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Niedbala, Angela Rochelle. "Kinetic studies of transcription initiation by wild type T7 RNA polymerase, his-tagged wild type T7 RNA polymerase and GP1-Lys222 T7 RNA polymerase." Thesis, Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/27288.

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Nottebaum, Sven. "In vitro assembly of recombinant archaeal RNA polymerases." Thesis, Imperial College London, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.443827.

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Ma, Hok-tsun, and 馬學俊. "RNA-Dependent RNA polymerase activity of the infectious bursal diseasevirus viral protein 1." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30408192.

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Curti, Elena. "Structure function studies of selected RNA and DNA polymerases." Thesis, University of Leeds, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414158.

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Yin, Chang. "Evolution of phage-type RNA polymerases in higher plants." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16270.

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In mono- und eudikotylen Pflanzen kodiert eine Genfamilie (RpoT, RNA-Polymerase des T3/T7-Typs) mitochondriale und plastidäre RNA-Polymerasen (RNAP), die den ungeraden T-Phagen-Polymerasen ähneln. RpoT-Gene von Angiospermen sind gut charakterisiert, während aus tiefer abzweigenden Pflanzenspecies bisher lediglich die Gene aus dem Moos Physcomitrella beschrieben wurden. Um einen Beitrag zur Aufklärung der molekularen Evolution der RpoT-Polymerasen im Pflanzenreich zu liefern und um Erkenntnisse über die potentielle Bedeutung von multiplen Phagen-Typ (RNAP) in Pflanzen zu gewinnen, wurden die RpoT-Gene aus dem Lycophyten Selaginella moellendorffii und aus dem basalen Angiosperm Nuphar advena identifiziert und charakterisiert. Selaginella moellendorffii (Moosfarn)-Trace-Sequenzdaten mit hoher Ähnlichkeit zu RpoT-Sequenzen von Angiospermen wurden benutzt, um das full-length SmRpoT-Gen und die entsprechende cDNA zu isolieren. Die SmRpoT-mRNA ist 3542 nt lang und weist einen offenen Leserahmen von 3006 nt auf, der für ein putatives Protein aus 1002 Aminosäuren mit einer molekularen Masse von 113 kDa kodiert. Das SmRpoT-Gen besteht aus 19 Exons und 18 Introns, die in ihren Positionen mit denen aus den Angiosperm- und Physcomitrella-Genen konserviert sind. Mittels Southernblot-Analyse wurde nachgewiesen, dass S. moellendorffii ein single-copy RpoT-Gen kodiert. Für das N-terminale Transitpeptid von SmRpoT konnte gezeigt werden, dass es bei transienter Expression in Arabidopsis- und Selaginella-Protoplasten den Transport von GFP (green fluorescent protein) exclusiv in Mitochondrien vermittelt. In N. advena wurden mittels Screening einer BAC-Bibliothek drei RpoT-Gene identifiziert. Sowohl die genomischen als auch die cDNA-Sequenzen wurden aufgeklärt. Die NaRpoT-mRNAs kodieren putative Polypeptide von 996, 990 und 985 Aminosären. Alle drei Gene besitzen 19 Exons und 18 Introns, die in ihren Positionen mit denen der RpoT-Gene aus Selaginella und allen anderen Landpflanzen konserviert sind. Die kodierten Proteine weisen auf Aminosäureebene einen hohen Konservierungsgrad auf, einschließlich aller essentiellen Regionen und Aminosäurereste, die für die T7-RNAP bekannt sind. Die N-terminalen Transitpeptide zweier der kodierten RNAP, NaRpoTm1 und NaRpoTm2, vermittelten den Import von GFP exclusiv in Mitochondrien, während die dritte Polymerase, NaRpoTp, in Chloroplasten importiert wurde. Interessanterweise muß die Translation der NaRpoTp-mRNA an einem CUG-Codon initiiert werden, um ein funktionelles Protein mit plastidärem Transitpeptid zu erhalten. Die N. advena RpoTp-RNAP ist somit neben AGAMOUS aus Arabidopsis und der RpoTp-RNAP aus Nicotiana, ein weiteres Beispiel für jene selten vorkommenden pflanzlichen mRNAs, deren Translation exclusiv an nicht-AUG-Codons initiiert wird. Die Rekonstruktion von phylogenetischen Bäumen resultierte in unterschiedlichen Positionen für die Selaginella- und Nuphar-Polymerasen: Im Gegensatz zu der RpoT-Polymerase aus S. moellendorffii und denen aus Physcomitrella, die in den phylogenetischen Analysen Schwesterpositionen zu allen anderen Phagentyp-RNAP der Angiospermen einnehmen, clusterten die Nuphar-RpoTs zusammen mit den deutlich separierten mitochondrialen (NaRpoTm1 und NaRpoTm2) und plastidären (NaRpoTp) Polymerasen. Selaginella kodiert eine einzige mitochondriale RNAP, während Nuphar zwei mitochondriale und eine plastidäre RNAP besitzt. Die Identifizierung einer Plastiden-lokalisierten Phagentyp-RNAP in diesem basalen Eudikotylen, die ortholog zu allen anderen RpoT-Enzymen der Blütenpflanzen ist, läßt darauf schließen, daß die Acquisition einer nukleär kodierten plastidären RNAP, die noch in den Lycopoden fehlt, nach der Trennung der Leucopoden von allen anderen Tracheophyten erfolgte. Eine “dual-targeting” RNAP (mitochondrial und plastidär lokalisiert), wie sie in Eudikotylen, nicht jedoch in Monokotylen vorkommt, wurde weder in Selaginella noch in Nuphar nachgewiesen, vermutlich ist sie ein evolutionäres Novum von eudikotylen Pflanzen wie Arabidopsis.
In mono- and eudicot plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species until recently were limited to the moss Physcomitrella. To elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we identified and characterized RpoT genes in the lycophyte Selaginella moellendorffii and the basal eudicot Nuphar advena. Selaginella moellendorffii (spikemoss) sequence trace data encoding a polypeptide highly similar to angiosperm and moss phage-type organelle RNA polymerases were used to isolate a BAC clone containing the full-length gene SmRpoT as well as the corresponding cDNA. The SmRpoT mRNA comprises 3452 nt with an open reading frame of 3,006 nt, encoding a putative protein of 1,002 amino acids with a molecular mass of 113 kDa. The SmRpoT gene comprises 19 exons and 18 introns, conserved in their position with those of the angiosperm and Physcomitrella RpoT genes. Using Southern blot analysis, it was shown that S. moellendorffii encodes a single RpoT gene. The N-terminal transit peptide of SmRpoT was shown to confer targeting of green fluorescent protein (GFP) exclusively to mitochondria after transient expression in Arabidopsis and Selaginella protoplasts. In Nuphar advena three RpoT genes were identified by BAC library screening. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those from S. moellendorffii and the RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of GFP exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp polymerase, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. Reconstruction of phylogenetic trees revealed different positions of the RpoTs from the lycophyte Selaginella and the basal eudicot Nuphar. In contrast to the RpoTs of S. moellendorffii and those of the moss Physcomitrella, which are according to the phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. Selaginella encodes a single mitochondrial RNAP, whereas Nuphar harbors two mitochondrial and one plastid phage-type polymerases. Identification of a plastid localized phage-type RNAP in this basal eudicot, orthologous to all other RpoTp enzymes of flowering plants, suggests that the acquisition of a nuclear encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastid RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar or Selaginella suggesting that its occurrence is an evolutionary novelty of eudicotyledoneous plants like Arabidopsis.

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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.

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The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans. In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation. Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes. This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.

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Phosiwa, Maanda Noaxe. "Molecular characterization of a porcine picobirnavirus RNA-dependent RNA polymerase." Diss., Pretoria : [s.n.], 2009. http://upetd.up.ac.za/thesis/available/etd-07152009-175205/.

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Swiatecka-Hagenbruch, Monika. "Phagenähnliche RNA-Polymerasen." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15924.

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Chloroplasten höherer Pflanzen haben kleine Genome. Trotzdem ist ihre Transkriptionsmaschinerie sehr komplex. Plastidäre Gene werden von plastidenkodierten (PEP) und kernkodierten RNA-Polymerasen (NEP) transkribiert. In der vorliegenden Arbeit wurden Promotoren plastidärer Gene und Operons von Arabidopsis thaliana charakterisiert. Zur Unterscheidung zwischen NEP- und PEP-Promotoren wurden erstmals spectinomycinbehandelte, chlorophylldefiziente Arabidopsis-Pflanzen mit fehlender PEP-Aktivität verwendet. Obwohl für einige Gene auch einzelne Promotoren lokalisiert wurden, wird die Transkription der meisten plastidären Gene und Operons an multiplen Promotoren initiiert. Der Vergleich plastidärer Promotoren von Tabak und Arabidopsis zeigte eine hohe Vielfältigkeit der Promotornutzung, die möglicherweise auch in anderen höheren Pflanzen vorkommt. Dabei stellt die individuelle Promotornutzung eine speziesspezifische Kontrollmöglichkeit der plastidären Genexpression dar. Das Kerngenom von Arabidopsis beinhaltet zwei Kandidatengene der NEP, RpoTp und RpoTmp, welche Phagentyp-RNA-Polymerasen kodieren. In der vorliegenden Arbeit wurde die Wirkung veränderter RpoTp-Aktivität auf die Nutzung von NEP- und PEP-Promotoren in transgenen Arabidopsis-Pflanzen mit verminderter und fehlender RpoTp-Aktivität untersucht. Im Keimlingsstadium konnten Unterschiede in der Promotornutzung zwischen Wildtyp und Mutanten beobachtet werden. Fast alle NEP-Promotoren wurden in Pflanzen mit verringerter oder fehlender RpoTp-Aktivität genutzt. Dabei zeigten nur einige von ihnen eine geringere Aktivität, andere wiederum waren sogar verstärkt aktiv. Der starke NEP-Promotor des essentiellen ycf1 Gens wurde in jungen Keimlingen ohne funktionelle RpoTp nicht genutzt. Die Ergebnisse zeigen, dass NEP gemeinsam von beiden Phagentyp-RNA-Polymerasen RpoTp und RpoTmp repräsentiert wird und dass beide sowohl eine überlappende, als auch eine spezifische Rolle in der Transkription plastidärer Gene innehaben.
Although chloroplasts of higher plants have small genomes, their transcription machinery is very complex. Plastid genes of higher plants are transcribed by the plastid-encoded plastid RNA polymerase PEP and the nuclear-encoded plastid RNA polymerases NEP. Here, promoters of plastid genes and operons have been characterized in Arabidopsis thaliana. For the first time spectinomycin-treated, chlorophyll-deficient Arabidopsis plants lacking PEP activity have been used to discriminate between NEP and PEP promoters. Although there are plastid genes that are transcribed from a single promoter, the transcription of plastid genes and operons by multiple promoters seems to be a common feature. Comparison of plastid promoters from tobacco and Arabidopsis revealed a high diversity, which my also apply to other plants. The diversity in individual promoter usage in different plants suggests that there are species-specific solutions for attaining control over gene expression in plastids. The nuclear genome of Arabidopsis contains two candidate genes for NEP transcription activity, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. In this study the usage of NEP and PEP promoters has been analysed in transgenic Arabidopsis plants with reduced and lacking RpoTp activity. Differences in promoter usage between wild type and mutant plants were most obvious early in development. Nearly all NEP promoters were active in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene was not transcribed in young seedlings without functional RpoTp. These results provide evidence for NEP being represented by two phage-type RNA polymerases RpoTp and RpoTmp that have overlapping as well as specific functions in the transcription of plastid genes.

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Bailey, Paul Austyn. "Inhibition of T7 RNA polymerase by T7 lysozyme." Diss., Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/30418.

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Clarke, Jane. "A kinetic study of the interaction of T7 RNA polymerase with its natural promoters." Thesis, Georgia Institute of Technology, 1990. http://hdl.handle.net/1853/25309.

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Lin, An-Chi. "Kinetic study of T7 RNA polymerase-promoter interactions on non-topologically constrained templates." Thesis, Georgia Institute of Technology, 1990. http://hdl.handle.net/1853/30697.

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鄭隆峰 and Lung-fung Cheng. "Modelling and sequence analysis of the collagen triple helix." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2001. http://hub.hku.hk/bib/B31969914.

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Yunnan, Jiang. "Testing the occurrence of forward hyper-translocation during the promoter escape transition / Jiang Yunnan." Connect to online version, 2009. http://ada.mtholyoke.edu/setr/websrc/pdfs/www/2009/381.pdf.

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Cheng, Lung-fung. "Modelling and sequence analysis of the collagen triple helix." Hong Kong : University of Hong Kong, 2001. http://sunzi.lib.hku.hk/hkuto/record.jsp?B2373615X.

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Fields, Scott. "Role of RNA polymerase I in maintaining the chromatin state of rRNA genes." Access citation, abstract and download form; downloadable file 4.79 Mb, 2004. http://wwwlib.umi.com/dissertations/fullcit/3131669.

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Xie, WenQin. "Molecular analysis of cyanobacterial RNA polymerase genes." Diss., Virginia Polytechnic Institute and State University, 1989. http://hdl.handle.net/10919/54816.

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The RNA polymerase genes rpoBC1C2 (the rpoB and rpoC2 are incomplete) of the cyanobacterium Nostoc commune have been cloned, sequenced, and expressed both in vivo and in vitro using E. coli systems. The rpo genes encode large subunits of DNA-dependent RNA polymerase. Two genes in N. commune, rpoC1 and rpoC2, correspond to rpoC of E. coli, which indicates a divergent evolution of RNA polymerase. The rpoBC1C2genes of Nostoc are linked in the order of rpoB, rpoC1, and rpoC2, and are transcribed differently from the corresponding rpo genes of E. coli. In E. coli the rpoBC genes are co-transcribed, together with two ribosomal protein genes. The Nostoc rpoB gene is transcribed by one promoter while the rpoC1C2 genes are co-transcribed by another promoter. Northern analysis of Nostoc RNA revealed two transcripts (3.1 and 5.6 kb), which were specific for the rpoB and rpoC1C2 genes, respectively. SDS-PAGE, Coomassie staining and immunoanalysis detected two polypeptides with molecular weights of 72 and 94 kd when the cloned rpoBC1C2 fragment was expressed in E. coli systems. These two polypeptides were assigned as products of rpoC1 and rpC2, respectively. The transcription of RNA polymerase genes of N. commune is sensitive to water stress (drying). The rpo transcription ceases upon drying and resumes after the dried cells have been rewetted for more than 5 min. The RNA polymerase enzyme itself, however, is stable under the same drying conditions.
Ph. D.

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Cozens, Christopher. "An adaptive path from DNA to RNA and ANA polymerases." Thesis, University of Cambridge, 2012. https://www.repository.cam.ac.uk/handle/1810/252281.

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Daly, Nicole Louise. "The deregulation of RNA polymerases I and III in tumours." Thesis, University of Glasgow, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400716.

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Swale, Christopher. "RNA binding and assembly of human influenza A virus polymerases." Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GREAV053/document.

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Le virus de la grippe A est un virus à ARN négatif appartenant à la famille des Orthomyxoviriadea dont la réplication se produit dans le noyau des cellules infectées. L'organisation du génome est segmentée en huit segments d'ARNv de polarité négative, codant pour un minimum de 16 protéines virales différentes. Ces ARN viraux (ARNv) sont en complexe avec de nombreuses copies de nucléoprotéines et liés par leurs extrémités 5' et 3' au complexe hétérotrimérique de l'ARN-polymérase ARN-dépendante composé des sous unités PA, PB1 et PB2. Cet assemblage macromoléculaire (ARNv / polymérase / NP) nommée Ribonucléoprotéine (RNP) constitue une entité génomique indépendante. Dans le contexte de la RNP, l'ARN-polymérase assure à la fois la transcription et la réplication du génome ARNv. En assurant ces deux fonctions, l'ARN-polymérase joue un rôle majeur dans la réplication virale et constitue une cible antivirale privilégiée. Les travaux de recherche présentés dans cette thèse se concentrent sur les éléments structuraux participants à l'assemblage de l'ARN polymérase et son interaction avec les avec les ARNv. Pour atteindre ces objectifs, notre laboratoire, en collaboration avec d'autres groupes, a mis en place un système d'expression en polyprotéines permettant d'exprimer la polymérase. Plus encore, cette méthode a aussi permis de reconstituer des complexes entre l'ARN-polymérase et des partenaires cellulaires, notamment RanBP5 qui appartient à la famille des importines-β
Influenza A virus is a negative-strand RNA virus belonging to the Orthomyxoviriadea family whose replication occurs in the nucleus of infected cells. The genome organisation of influenza virus is segmented in eight vRNA segments of negative polarity coding for at least 16 different viral proteins. Each vRNA is bound to multiple copies of nucleoprotein (NP) and to the heterotrimeric RNA-dependent RNA-polymerase complex (PA, PB1 and PB2) through its 5' and 3' extremities. This macromolecular assembly (vRNA/polymerase/NP) forms the ribonucleoprotein (RNP) particle, which acts as a separate genomic entity within the virion. The RNP complex is at the core of viral replication and in the context of RNPs, the polymerase performs both transcription and replication of the vRNA genome. As such, the polymerase constitutes a major antiviral drug target. The research work presented within this thesis focuses on the underlying determinants of the RNA polymerase assembly process and its interaction with its vRNA genome. To fulfill these goals, our lab, in collaboration with other groups, has set up a novel polyprotein expression system to express the polymerase but also to reconstitute polymerase and cellular partner complexes, notably RanBP5, which belongs to the importin-β family

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Minaker, Sean Wilson. "Defects in messenger RNA processing and biogenesis of RNA polymerases contribute to eukaryotic genome instability." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44216.

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Genome instability has been observed in mutants involved in various aspects of transcription and RNA processing. The prevalence of this mechanism among essential chromosome instability (CIN) genes remains unclear. In this thesis, it is shown that RNA biogenesis mutants exhibit elevated sensitivity to DNA damaging agents. A secondary screen for increased Rad52 foci in CIN mutants, representing ~25% of essential genes, identified seven essential subunits of the mRNA cleavage and polyadenylation (mCP) machinery. Genome-wide analysis of fragile sites by ChIP-chip of phosphorylated-H2A in these mutants supported a transcription-dependent mechanism of DNA damage characteristic of RNA:DNA hybrids known as R-loops, which were subsequently observed in mCP mutants. Among the CIN mutants with elevated Rad52 foci levels were the GPN proteins, a poorly-characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2 and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here, it is shown that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1, and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition and defects in RNA polymerase II nuclear localization. Importantly, previously unrecognized RNA polymerase III localization defects were observed in GPN2, GPN3 and IWR1 mutant backgrounds but no localization defects of unrelated nuclear proteins or of RNA polymerase I were found. In this study, it was shown that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1 suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common function in RNA polymerase assembly and subsequent transport. These findings demonstrate how mRNA cleavage and polyadenylation and proper RNA polymerase assembly contribute to maintenance of genome integrity and may be relevant to certain human cancers.

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25

Nayak, Dhananjaya. "Conformational mechanisms in T7 RNA polymerase transcription a dissertation /." San Antonio : UTHSC, 2008. http://learningobjects.library.uthscsa.edu/cdm4/item_viewer.php?CISOROOT=/theses&CISOPTR=44&CISOBOX=1&REC=11.

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26

Armache, Karim-Jean. "Crystal structures of the complete 12-subunit RNA polymerase II and its subcomplex Rpb4-7, and modeling of RNA polymerases I and III." [S.l.] : [s.n.], 2005. http://edoc.ub.uni-muenchen.de/archive/00004915.

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27

Armache, Karim. "Crystal structures of the complete 12-subunit RNA polymerase II and its subcomplex Rpb4/7, and modeling of RNA polymerases I and III." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-49156.

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28

Mischo, Hannah. "Disengaging Polymerases : Transcriptional termination by RNA polymerase II in Saccharomyces cerevisiae and the maintenance of genome integrity." Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.514968.

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29

Luo, Jie. "The evolution and composition of RNA polymerase IV in plants /." Thesis, Connect to this title online; UW restricted, 2006. http://hdl.handle.net/1773/5190.

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30

Jones, Louisa Alice School of Biotechnology And Biomolecular Sciences UNSW. "Aptamers to the hepatitis C virus polymerase." Awarded by:University of New South Wales. School of Biotechnology And Biomolecular Sciences, 2005. http://handle.unsw.edu.au/1959.4/32734.

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Treatments for the hepatitis C virus (HCV) are currently only partially effective. Research into antivirals directed at HCV viral proteins are commonly based and tested on a single genotype, namely genotype 1. This is despite the high level of variability of the RNA virus and the frequency of infection with genotypes other than 1. The systematic evolution of ligands by exponential enrichment (SELEX) is a novel in vitro approach for the isolation of antiviral agents. SELEX allows rapid screening of vast nucleic acid libraries to isolate sequences (termed aptamers) that bind to target proteins with high affinity. The SELEX approach was used in the present study to isolate DNA aptamers to the RNAdependent RNA polymerase (RdRp) [non-structural protein B (NS5B)] protein of HCV subtype 3a, with the aim of inhibiting polymerase activity. Ten rounds of selection were performed using a Biacore 2000 and resultant aptamers cloned from rounds 2, 4, 8 and 10. Sequences of aptamers were aligned to elucidate common motifs and a proportion of the aptamers from rounds 8 and 10 (29/48) were screened for binding ability using the Biacore. The five ???best binding??? aptamers were investigated for inhibition of 3a polymerase activity in an in vitro polymerase assay. Two aptamers, r10/43 and r10/47, were chosen for further studies based on their ability to inhibit polymerase activity. The inhibition constants (Ki) of r10/43 and r10/47 were estimated to be 1.4 + 2.4 nM and 6.0 + 2.3 nM respectively. The affinity (Kd) of these aptamers for the 3a polymerase was estimated to be 1.3 + 0.3 nM (r10/43) and 23.5 + 6.7 nM (r10/47). The estimated inhibition and dissociation constants of these two aptamers are among the best for inhibitory aptamers of the HCV enzymes (polymerase and protease). Inhibition of HCV 3a polymerase appeared to be specific for r10/47, whilst r10/43 also had some inhibitory effect on norovirus and ??6 polymerase activity. This study is the first description of an inhibitor to the HCV subtype 3a polymerase that investigates genotypic specificity of targeted antivirals.

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31

Firth, Andrew Graeme. "Synthesis and Characterisation of Fluorescent RibonucleotideSubstrates for DNA Dependent RNA Polymerases." Thesis, University of York, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507614.

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32

Chang, Mingi. "Analysis of autoantibodies against RNA polymerases in patients with systemic sclerosis /." free to MU campus, to others for purchase, 1998. http://wwwlib.umi.com/cr/mo/fullcit?p9924872.

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33

Adams, Jonathan Weldon. "Kinetic assay of T7 activity on mutant promoters : method development and experimental design." Thesis, Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/29908.

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34

Sheagley, Eric Eugene. "Mechanisms of transcription elongation and the nuclease activity of RNA polymerase II /." view abstract or download file of text, 2003. http://wwwlib.umi.com/cr/uoregon/fullcit?p3080598.

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Thesis (Ph. D.)--University of Oregon, 2003.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 119-129). Also available for download via the World Wide Web; free to University of Oregon users.

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35

Moreland, Rodney J. "Molecular interactions in RNA polymerase II and III transcription systems /." Full-text version available from OU Domain via ProQuest Digital Dissertations, 1998.

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36

Chin, Wing-hong, and 錢永康. "Polymerase activity of chimeric polymerase : a determining factor for an influenza virus to be a pandemic strain." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/193398.

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The influenza polymerase is a complex of three subunits, polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1) and polymerase acidic protein (PA). It associates with the viral RNA segment and nucleoprotein (NP) to form a viral ribonucleoprotein (vRNP) complex which is important for transcription and replication of the viral genome. Concurrently, the previous three influenza pandemics viruses contain reassorted vRNP of different origins. This leads to the aim of study to investigate the role of polymerase in the pandemic viruses. By reconstitution of vRNPs in human cells, it was demonstrated that vRNPs of H2N2 and H3N2 pandemic viruses had higher polymerase activity than the H2N2 seasonal viruses in-between them. The recombinant virus with H2N2 pandemic vRNP also showed faster growth kinetics in the early stage of viral replication and better adaptability to the selective environment with neuraminidase inhibitor than the recombinant virus with H2N2 seasonal vRNP, which had a lower polymerase activity. Reconstitution of chimeric vRNPs of H2N2 pandemic and seasonal viruses revealed that PB2, PB1 and PA were responsible for the difference in polymerase activity between them. Five residues, one in PB2, three in PB1 and one in PA were identified to be significant for the polymerase activity change. These polymerase subunits and residues may act as part of the determining factors for the H2N2 pandemic virus. Furthermore, PB2-627 has been shown to have stringent host specificity and affect polymerase activity and viral replication. Recombinant viruses in mammalian and avian cells with random mutation were generated at this position. It showed that the amino acids at this position are not restricted to those appear in the nature for generating viable viruses. It was also observed that the avian-derived viruses generally had lower polymerase activity and reduced growth kinetics in mammalian cells, while part of the mammalian-derived viruses had lower polymerase activity and reduced growth kinetics in avian cells. This consolidated the role of PB2-627 on host specificity and demonstrated the possibility of some novel amino acids for this position, which may play a role in the future influenza pandemic. The 2009 H1N1 pandemic virus contains a reassorted vRNP with subunits of avian, human and swine origins. This prompts me to compare the polymerase activity of all the 81 possible combinations of chimeric vRNPs of three different origins. The results were statistically analyzed and several single subunit factors and interactions between vRNP subunits were identified to significantly affect the polymerase activity. In order to reduce the effort and resources required, a fractional factorial design of 27 experimental runs was developed to substitute the 81-combination full factorial design for identifying the significant single subunit factors that affect the polymerase activity. Overall, this study identified some factors that may contribute to a pandemic virus and allows us to have better understanding of the role of polymerase in a pandemic virus. These findings may contribute to evaluating the pandemic potential of the novel virus that emerges or may emerge in the nature and enhances the preparedness towards the next pandemic influenza.
published_or_final_version
Public Health
Doctoral
Doctor of Philosophy

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37

Francis, Rawle Friedman Simon H. "Inhibition of human telomerase by targeting its transitory RNA/DNA heteroduplex." Diss., UMK access, 2005.

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Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2005.
"A dissertation in pharmaceutical sciences and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed June 23, 2006; title from "catalog record" of the print edition. Includes bibliographical references (leaves 327-353). Online version of the print edition.

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38

Li, Tin-wai Olive. "Influenza polymerase subunit compatibility between human H1 and H5 viruses." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B41896890.

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39

Kantidakis, Theodoros. "In vivo studies of repressors of RNA polymerase III transcription." Thesis, Thesis restricted. Connect to e-thesis to view abstract, 2008. http://theses.gla.ac.uk/161/.

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Thesis (Ph.D.) - University of Glasgow, 2008.
Ph.D. thesis submitted to the Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, 2008. Includes bibliographical references. Print version also available.

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40

Xiong, Yalin. "Downstream NTP effects on human RNA polymerase II transcription elongation." Diss., Connect to online resource - MSU authorized users, 2008.

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Thesis (Ph.D.)--Michigan State University. Dept. of Biochemistry and Molecular Biology, 2008.
Title from PDF t.p. (viewed on Apr. 2, 2009) Includes bibliographical references. Also issued in print.

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41

Hemming, Sally Anne. "Functional analysis of the ninth subunit of yeast RNA polymerase II, RPB9 / by Sally Anne Hemming." *McMaster only, 1998.

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42

Warshamana, Gnana Sakuntala. "Interactions of T7 RNA polymerase with its promoters : Part I: T7 promoter contacts essential for promoter activity in vivo ; Part II: Isolation and characterization of a mutant T7 RNA polymerase with altered promoter specificity." Diss., Georgia Institute of Technology, 1992. http://hdl.handle.net/1853/26303.

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43

Henfrey, R. D. "In vitro transcription of exogenous plant DNA." Thesis, University of Hertfordshire, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381604.

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44

Stevenson, Abigail Louise. "Cytoplasmic polyadenylation in S. pombe." Thesis, University of Oxford, 2005. http://ora.ox.ac.uk/objects/uuid:4ef2e3ad-8bac-44b5-b838-4023b18c4693.

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Cid1 is a cytoplasmic member of a novel class of regulatory poly(A) polymerases discovered recently in yeast, worms and vertebrates. Previous genetic studies in the fission yeast, Schizosaccharomyces pombe, suggested a role for Cid1 in the checkpoint response to replication stress, but it was not known how a poly(A) polymerase might contribute to this response. Further investigations into the mode of action of Cid1 were therefore undertaken in this study. Cid1 is likely to target specific RNAs for polyadenylation; potential RNA substrates were identified using the complementary methods of microarray hybridisation and whole proteome analysis using two-dimensional liquid chromatography. These experiments revealed that Cid1 does not affect RNAs during normal, unperturbed growth but instead alters the expression of specific subsets of genes during replication stress. Many RNAs affected by Cid1 in these circumstances were cell-cycle dependent and telomeric transcripts, including those encoding histones and a novel RecQ helicase, Rqh2. As Cid1 lacks an RNA recognition motif, it is unlikely to bind selectively to RNA targets on its own. Cid1-interacting proteins were identified using yeast two-hybrid and tandem affinity purification methods. From these studies, novel members of a Cid1 complex have been discovered including: a previously uncharacterised metallo-beta-lactamase, RNA-binding proteins, ribosomal proteins and a telomere-binding protein. Together, these approaches are leading to a model for the role of cytoplasmic polyadenylation by Cid1 in checkpoint control.

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45

Zechner, Kerstin. "3' end processing and RNA polymerase II transcription termination in protein coding genes in the nematode C. elegans." Thesis, University of Oxford, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.564397.

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In all organisms studied so far, the recognition of a functional poly(A) site is essential for RNA polymerase II termination at the end of nearly all genes transcribed by this enzyme (Whitelaw and Proudfoot, 1986; Guo et al., 1995; Birse et al. 1997). A number of eukaryotes have some of their genes organised in polycistronic structures which resemble bacterial operons (Davis and Hodgson, 1997; Ganot et al., 2004; Spieth et al. 1993), and in C. elegans, approximately 20% of all genes are contained within these operon-like structures (Blumenthal et al., 2002). Here, functional poly(A) sites will be synthesised and recognised by RNA polymerase II at the end of each gene within the operon, however termination of the polymerase only occurs at the final gene of the polycistronic transcription unit In these studies, we analyse the halting of RN A polymerase II transcription at the end of monocistronic genes and furthermore observe how premature RNA polymerase II termination is prevented during polycistronic transcription in the nematode C. elegans. We predominantly make use of reverse transcriptase PCR-based techniques to examine these mechanisms. We show that a large increase in pre-mRNAs stretching into the 3' flank of genes can be detected in worms depleted of the riboexonuclease XRN-2, indicating that this enzyme may have a possible role in RNA pol II termination and 3' end formation in C. elegans. Furthermore, we provide evidence that the polymerase can read into telomeric structures in the nematode. Also, we demonstrate that an RNAi-mediated knockdown of the UI-70K subunit of the UI snRNP causes a drop in polycistronic transcripts, providing a link between cis- splicing and the prevention of premature RNA polymerase II termination at operon-internal poly(A) sites. Finally, we illustrate that operon-internal poly(A) sites are capable of directing efficient 3' end formation outside of a polycistronic background. Together, these findings provide valuable insights into the mechanisms involved in directing or preventing premature RNA polymerase II transcription termination at C. elegans poly(A) sites.

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46

Martinez, Maria Juanita. "Transcription factor IIIB binding to two classes of Alanine tRNA gene promoters of the silkmoth, Bombyx mori /." view abstract or download file of text, 2001. http://wwwlib.umi.com/cr/uoregon/fullcit?p3018382.

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Thesis (Ph. D.)--University of Oregon, 2001.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 128-143). Also available for download via the World Wide Web; free to University of Oregon users.

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47

Chisholm, Robert David. "Mutations in RNA polymerase II that affect poly (a)-dependent termination /." view abstract or download file of text, 2006. http://proquest.umi.com/pqdweb?did=1188876151&sid=1&Fmt=2&clientId=11238&RQT=309&VName=PQD.

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Thesis (Ph. D.)--University of Oregon, 2006.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 80-86). Also available for download via the World Wide Web; free to University of Oregon users.

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48

Li, Tin-wai Olive, and 李天慧. "Influenza polymerase subunit compatibility between human H1 and H5 viruses." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B41896890.

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49

rob, abdur. "Protein-Nucleic Acid Interactions in Nuclease and Polymerases." Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/chemistry_diss/54.

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DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro. In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but not rNTP. We have also observed that many other DNA polymerases have this type of the RNA-dependent pyrophosphatase activity. Our experimental data suggest that the exonuclease active sites most likely play the critical role in this RNA-dependent dNTP hydrolysis, which might have a broader impact on biological systems. On the basis of the crystal structure of a ternary complex of RNase H (Bacillus halodurans), DNA, and RNA, we have introduced the selenium modification at the 6-position of guanine (G) by replacing the oxygen (SeG). The SeG has been incorporated into DNA (6 nt. - 6 nucleotides) by solid phase synthesis. The crystal structure and biochemical studies with the modified SeG-DNA indicate that the SeDNA can base-pair with the RNA substrate and serve as a template for the RNA hydrolysis. In the crystal structure, it has been observed that the selenium introduction causes shifting (or unwinding) of the G-C base pair by 0.3 Å. Furthermore, the Se-modification can significately enhance the phosphate backbone cleavage (over 1000 fold) of the RNA substrate, although the modifications are remotely located on the DNA bases. This enhancement in the catalytic step is probably attributed to the unwinding of the local duplex, which shifts scissile phosphate bond towards the enzyme active site. Our structural, kinetic and thermodynamic investigations suggest a novel mechanism of RNase H catalysis, which was revealed by the atom-specific selenium modification.

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50

Nesser, Nicole Katherine. "RNA polymerase II subunit RPB9 is important for transcriptional fidelity and processivity /." view abstract or download file of text, 2005. http://wwwlib.umi.com/cr/uoregon/fullcit?p3201694.

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Thesis (Ph. D.)--University of Oregon, 2005.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 78 - 83). Also available for download via the World Wide Web; free to University of Oregon users.

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Dissertations / Theses on the topic 'RNA polymerases'

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