Dissertations / Theses on the topic 'Transcription mechanism'
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1
An, Sungwhan. "Mechanism of coronavirus transcription /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.
2
Ng, King Pan. "The mechanism of the transcription activation mediated by the Ewing sarcoma activation domain /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202008%20NG.
3
Churcher, Mark Jonathan. "Studies on the mechanism of action of Tat." Thesis, Open University, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.359975.
4
Ignatov, Michael E. "Cis-Acting Elements in Mechanism of HIV-1 Reverse Transcription." Case Western Reserve University School of Graduate Studies / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=case1149088883.
5
Grably, Melanie R. "Revealing the mechanism of HSP104 transcription initiation in the yeast S.cerevisiae." E-thesis Full text, 2008. http://shemer.mslib.huji.ac.il/dissertations/W/JSL/001444203.pdf.
6
Cocklin, Simon. "Investigation into the molecular mechanism of nitrogen metabolite repression." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327280.
7
Hegde, Nagaratna Shridhar. "Investigating the molecular mechanism of thiostrepton inhibition of FOXM1 activity." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609983.
8
Shakya, Arvind. "Mechanism of matrix metalloproteinase expression in atherosclerosis /." Free to MU Campus, others may purchase, 2003. http://wwwlib.umi.com/cr/mo/fullcit?p1418063.
9
Mukherjee, Pooja. "Study of the co-translational assembly mechanism of transcription complexes in mammalian cells." Thesis, Strasbourg, 2019. http://www.theses.fr/2019STRAJ051.
Abstract:
La majorité des processus biologiques sont réalisés par des complexes protéiques multisubunités dans les cellules et une quantité importante d'énergie est requise par les cellules pour construire ces énormes complexes. Contrairement aux bactéries, les gènes codant pour les protéines sont dispersés dans le génome des eucaryotes, ce qui complique la compréhension de l'assemblage des complexes protéiques. En utilisant l'immunoprécipitation d'ARN suivie par la détection des ARNm à l'échelle du génome par analyse par micropuce, ARN molécule unique, FISH, immunofluorescence, cellules souches embryonnaires knock-out de souris et approches de permutation de domaines, nous montrons que les complexes de transcription multisubunit de mammifère s'assemblent de manière co-traductionnelle. Nous démontrons que les domaines de dimérisation et leurs positions dans les sous-unités en interaction déterminent la voie d'assemblage de co-traduction (simultanée ou séquentielle). En outre, les expériences cytoplasmiques IF-smFISH et bicolores smFISH indiquent que l'assemblage de co-traduction décrit se produit clairement dans le cytoplasme de cellules humaines. Des résultats identiques dans les cellules de levure, de souris et humaine suggèrent que l'assemblage par co-traduction est un mécanisme général chez les eucaryotes, qui pourrait être nécessaire pour éviter les interactions non spécifiques et l'agrégation de protéines dans la celluleMajority of the biological processes are carried out by multisubunit protein complexes in cells and a significant amount of energy is required by the cells to build these huge complexes. Unlike bacteria, genes encoding proteins are dispersed in the genome of eukaryotes and this makes the assembly of protein complexes more complicated to understand. By using RNA immunoprecipitation followed by genome-wide detection of mRNAs by microarray analysis, single molecule RNA FISH, immunofluoresence, mouse knock-out embryonic stem cells and domain swapping approaches, we show that the mammalian multisubunit transcription complexes assemble co-translationally. We demonstrate that the dimerization domains and their positions in the interacting subunits determine the co-translational assembly pathway (simultaneous or sequential). Furthermore, cytoplasmic IF-smFISH and two-colour smFISH experiments indicate that the described co-translational assembly is clearly occurring in the cytoplasm of human cells. Identical results in yeast, mouse and human cells suggests that co-translational assembly is a general mechanism in eukaryotes which might be necessary to avoid non-specific interactions and protein aggregation in the cell
10
Rowe-Magnus, Dean Allistair. "The mechanism of transcription activation by the Bacillus subtilis response regulator, Spo0A." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape7/PQDD_0022/NQ38968.pdf.
11
Hieb, Aaron R. "Architecture and mechanism of the human RNA polymerase II general transcription machinery." Diss., Connect to online resource, 2006. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3219003.
12
Wijayatilake, Hashanthi. "Insights into the mechanism and function of transcription-dependent intra-genic looping." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510400.
13
Hughes, Amanda Dawn. "Mechanism of enhancer-dependent transcription in Escherichia coli by σâ¿-RNA polymerase." Thesis, University of York, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.399583.
14
Abeysinghe, Arachchige Jayami Kaushalya Abeysinghe. "Mechanism of WRKY transcription factors-mediated defense and heterosis in Arabidopsis polyploids." HKBU Institutional Repository, 2018. https://repository.hkbu.edu.hk/etd_oa/596.
Abstract:
WRKY transcription factors (TFs) belong to a large family of regulatory proteins in plants that modulate many plant processes. Extensive studies have been conducted on WRKY-mediated defense response in Arabidopsis thaliana and many crop species. This study aims to investigate the potential roles and contributions of WRKY TFs regulation in improving defense response in the resynthesized Arabidopsis allotetraploids (Arabidopsis suecica) from two related autotetraploid progenitors, Arabidopsis thaliana (At4) and Arabidopsis arenosa (Aa). Upon infection by Pseudomonas syringae (Pst), the allotetraploids has showed enhanced resistance against the pathogen when compared to the parents. Rapid induction of WRKY18, WRKY40, WRKY38, WRKY53, WRKY6; MAP kinase pathway related genes, WRKY33, PAD3; SA-pathway related genes, ICS1, EDS1, PBS3, MYB31; was evident in response to Pst and salicylic acid treatment in the allotetraploids. Cleaved amplified polymorphic sequences analysis further revealed that the AtWRKY18, AaWRKY40, AtWRKY33, and AtWRKY60 alleles expressed at higher levels when compared to their respective homoeologs in the allotetraploids, suggesting potential altered protein-protein interaction networks in the hybrids. Therefore, a split-luciferase complementation assay was used to characterize and quantify protein-protein interaction among these homoeologous WRKYs in the allotetraploids. Results showed that preferential protein-protein interactions exist for the cis-interacting AtWRKY18/AtWRKY18 homodimer or trans-interacting AtWRKY18/AaWRKY40 heterodimer when compared to the respective interacting complexes. In addition, differential affinities of WRKY18 and WRKY40 homo- and hetero- dimers toward the W-boxes at the WRKY60 promoter were observed. In the allotetraploids, PR1 expression was repressed under basal state when compared to the progenitors. Although PR1 is expressed at a higher level in A. thaliana, its expression fold change was higher and faster in the all otetraploids upon salicylic acid treatment. Transient expression of WRKY18 or WRKY40 homodimer in various combinations induced differential expression of PR1 gene in their respective wrky18 and wrky40 Arabidopsis thaliana mutants. In contrast, similar PR1 induction by homodimer in various combinations was observed when they were transiently expressed in the allotetraploids. In addition, transgenic AtWRKY18 overexpression plant displayed enhanced disease resistance against Pst when compared to AaWRKY18 overexpression lines. Such enhanced disease resistance was found to associate with the higher expression of PR1 and PR2 in AtWRKY18 transgenic lines. Moreover, differential Pst-induced expression of the direct targets (ICS1, EDS1 and PBS3) of WRKY18 in the Arabidopsis AtWRKY18 and AaWRKY18 overexpressors supported a biological difference between the At and Aa homodimers in mediating the targets regulation, thus contributing to the difference in disease responses. Overall, our findings suggested that the rapid differential alleles expression and altered protein-protein or protein-DNA interactions of WRKY transcription factors could contribute to the improved defense in the allotetraploids, providing a molecular basis of for heterotic phenotype development in hybrids.
15
Wu, Xuebing Ph D. Massachusetts Institute of Technology. "The mechanism and function of pervasive noncoding transcription in the mammalian genome." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/93042.
Abstract:
Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014.Cataloged from PDF version of thesis.
Includes bibliographical references
The vast majority of the mammalian genome does not encode proteins. Only 2% of the genome is exonic, yet recent deep survey of human transcripitome suggested that 75% of the genome is transcribed, including half of the intergenic regions. Such pervasive transcription typically leads to short-lived, low-copy number noncoding RNAs (ncRNAs). We are starting to understand the biogenesis and mechanisms regulating the noncoding transcription. However, it is still unclear what's the functional impact of pervasive transcription and the ncRNAs at the level of the'genome, the cell, and the organism. A large fraction of ncRNAs in cells is generated by divergent transcription that occurs at the majority of mammalian gene promoters. RNA polymerases transcribe divergently on opposite strands, producing precursor mRNAs (pre-mRNAs) on one side and promoter upstream antisense RNAs (uaRNAs) on the other side. Like typical products of pervasive transcription, uaRNAs are relatively short and unstable as compared to pre-mRNAs, suggesting there are mechanisms suppressing uaRNA transcription and enforcing promoter directionality. We describe the Ul-PAS axis, a mechanism that enhances gene transcription but suppresses noncoding transcription. Two RNA processing signals, the Ul signal, or 5' splice site sequences recognized by Ul snRNP during splicing, and polyadenylation signal (PAS), differentially mark the two sides of gene transcription start site (TSS), ensuring the generation of full-length mRNA but inducing early termination of uaRNAs. The Ul-PAS axis also suppresses pervasive transcription on the antisense strand of genes, as well as intergenic transcription. Transcription is a mutagenic process that could accelerate evolution. We uncover a link between pervasive transcription and genome evolution. Specifically, transcription-induced mutational bias in germ cells could strengthen the Ul-PAS axis, which in turn enhances transcription, thus forming a positive feedback loop, which eventually drives new gene origination, and facilitates genome rearrangements. Tools to directly interfere with transcription with specificity are necessary to understand the function of noncoding transcription, especially when the RNA product is rapidly degraded or nonfunctional. The newly emerged CRISPR-Cas9 system provides the opportunity to target any desired locus. We comprehensively characterize the binding specificity of Cas9 in the mouse genome. We find that Cas9 specificity varies dramatically but in a predictable manner, depending on the seed sequence and chromatin accessibility. Our results will facilitate Cas9 target design and enable genome manipulation with high precision.
by Xuebing Wu.
Ph. D.
16
Chakraborty, Atanu. "Mechanism Of mom Gene Transactivation By Transcription Factor C Of Phage MU." Thesis, Indian Institute of Science, 2006. https://etd.iisc.ac.in/handle/2005/275.
Abstract:
Regulation of transcription initiation is the major determining event employed by the cell to control gene expression and subsequent cellular processes. The weak promoters, with low basal transcription activities, are activated by activators. Bacteriophage Mu mom gene, which encodes a unique DNA modification function, is detrimental to cell when expressed early or in large quantities. Mu has designed a complex, well-controlled and orchestrated regulatory network for mom expression to ensure its synthesis only in late lytic cycle. The phage encoded transcription activator protein C activates the gene by promoter unwinding of the DNA and thereby recruiting of RNAP to the promoter.
C protein functions as a dimer for DNA binding and transcription activation. Mutagenesis and chemical crosslinking studies revealed that the leucine zipper motif, and not the coiled coil motif in the N terminal region, is responsible for C dimerization. The DNA binding domain of C is a HTH domain which is preceded by the leucine zipper motif. The C protein is one of the few examples in the bacterial proteins containing both leucine zipper and HTH domain.
Most of the transcription activators either influence initial binding of RNAP or conversion of closed to open complex formation. Very few activators act at subsequent steps of promoter-polymerase interaction. Earlier studies showed high level of transcription from a mutant mom promoter, tin7. Addition of C further increased transcription from Ptin7 indicating that C may have a role beyond polymerase recruitment. Each steps of transcription initiation have been dissected using the Ptin7 and a positive control (pc) mutant of C, R105D. The results revealed multi-step transcription activation mechanism for C protein at Pmom. C recruits RNAP at Pmom and subsequently increases the productive RNAP-promoter complex and enhances promoter clearance.
To further understand the C mediated transactivation mechanism, interaction between C and RNAP was assessed. C interacts with holo and core RNAP only in presence of DNA. Positive control mutants of C, F95A and R015D, were found to be compromised in RNAP interactions. These mutants were efficient in RNAP recruitment to Pmom but do not enhance promoter clearance. Trypsin cleavage protection experiment indicated that probably C protein interacts with b¢ subunit of RNAP. Interaction between C and RNAP appears to enhance the formation of productive RNAP-promoter complex leading to promoter clearance.
The connection between activator-polymerase interaction and transcription activation is well documented where the recruitment of RNAP is influenced. In case of activators acting at post recruitment steps of initiation, the role of polymerase contact is poorly understood. Our study shows that activator-polymerase interaction can lead to increased promoter clearance at Pmom by overcoming abortive initiation.
17
Chakraborty, Atanu. "Mechanism Of mom Gene Transactivation By Transcription Factor C Of Phage MU." Thesis, Indian Institute of Science, 2006. http://hdl.handle.net/2005/275.
Abstract:
Regulation of transcription initiation is the major determining event employed by the cell to control gene expression and subsequent cellular processes. The weak promoters, with low basal transcription activities, are activated by activators. Bacteriophage Mu mom gene, which encodes a unique DNA modification function, is detrimental to cell when expressed early or in large quantities. Mu has designed a complex, well-controlled and orchestrated regulatory network for mom expression to ensure its synthesis only in late lytic cycle. The phage encoded transcription activator protein C activates the gene by promoter unwinding of the DNA and thereby recruiting of RNAP to the promoter.
C protein functions as a dimer for DNA binding and transcription activation. Mutagenesis and chemical crosslinking studies revealed that the leucine zipper motif, and not the coiled coil motif in the N terminal region, is responsible for C dimerization. The DNA binding domain of C is a HTH domain which is preceded by the leucine zipper motif. The C protein is one of the few examples in the bacterial proteins containing both leucine zipper and HTH domain.
Most of the transcription activators either influence initial binding of RNAP or conversion of closed to open complex formation. Very few activators act at subsequent steps of promoter-polymerase interaction. Earlier studies showed high level of transcription from a mutant mom promoter, tin7. Addition of C further increased transcription from Ptin7 indicating that C may have a role beyond polymerase recruitment. Each steps of transcription initiation have been dissected using the Ptin7 and a positive control (pc) mutant of C, R105D. The results revealed multi-step transcription activation mechanism for C protein at Pmom. C recruits RNAP at Pmom and subsequently increases the productive RNAP-promoter complex and enhances promoter clearance.
To further understand the C mediated transactivation mechanism, interaction between C and RNAP was assessed. C interacts with holo and core RNAP only in presence of DNA. Positive control mutants of C, F95A and R015D, were found to be compromised in RNAP interactions. These mutants were efficient in RNAP recruitment to Pmom but do not enhance promoter clearance. Trypsin cleavage protection experiment indicated that probably C protein interacts with b¢ subunit of RNAP. Interaction between C and RNAP appears to enhance the formation of productive RNAP-promoter complex leading to promoter clearance.
The connection between activator-polymerase interaction and transcription activation is well documented where the recruitment of RNAP is influenced. In case of activators acting at post recruitment steps of initiation, the role of polymerase contact is poorly understood. Our study shows that activator-polymerase interaction can lead to increased promoter clearance at Pmom by overcoming abortive initiation.
18
Shakya, Arvind. "Mechanism of matrix metalloproteinase-14 (mmp-14) regulation during atherosclerosis." Diss., Columbia, Mo. : University of Missouri-Columbia, 2006. http://hdl.handle.net/10355/4436.
Abstract:
Thesis (Ph. D.)--University of Missouri-Columbia, 2006."December 2006" The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. Includes bibliographical references.
19
Davidson, Alexander F. "Elucidating the mechanism of localised mDNA translation during Drosophila oogenesis." Thesis, University of Oxford, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711933.
20
Préfontaine, Gratien G. "Selective binding of steroid receptors to octamer transcription factors determines transcriptional synergism at the mouse mammary tumor virus promoter: A molecular mechanism for transcription factor recruitment to promoter DNA." Thesis, University of Ottawa (Canada), 2001. http://hdl.handle.net/10393/9111.
Abstract:
The glucocorticoid receptor (GR) and octamer transcription factors -1 and -2 (Oct -1/-2) function synergistically to activate gene transcription from the Mouse Mammary Tumor Virus (MMTV) promoter. Mechanisms responsible for the transcriptional synergy have not been characterized. I demonstrated a protein-protein interaction between rat GR and human Oct-1/-2 in vivo, and showed the interaction was sensitive to GR point mutations C500Y and L501P. This interaction correlated with the recruitment of Oct-l/-2 to promoter DNA and appeared to contribute at least in part to the transcriptional synergy observed. Based on this observation, a molecular mechanism was proposed that would be expected to restrict gene transcription to regulatory regions containing binding sites for both factors. The direct protein-protein interaction with GR and Oct-1/-2 mapped to the Octamer factor homeodomains suggesting the potential for a broadly based interaction for homeodomain proteins. Previously, in vitro binding studies had identified several nuclear hormone receptors with the potential to bind to the POU domain of octamer factors. However in vivo, only the GR, the progesterone receptor (PR) and the androgen receptor (AR) appeared to have the potential to interact with octamer factors physically and functionally through their DNA-binding and hinge domains. In contrast, the mineralocorticoid receptor (MR) failed to interact. These steroid receptors can activate transcription through common hormone response elements (HREs) but they perform distinct physiological functions by regulating unique target genes. In transient transfection assays, I demonstrated that these steroid receptors could activate transcription from the MMTV promoter to similar levels. However, differential modes of gene regulation were employed by individual steroid receptors. Transcription mediated by GR and PR was dependent on the octamer motifs while that mediated by MR and AR was octamer motif independent. The configuration of the MMTV HREs was restricted to GR- and PR-mediated transcription but octamer factor recruitment to DNA permitted gene transcription from the MMTV promoter. These results suggest that the configuration of the HREs on the MMTV promoter determine steroid receptor-specific transcriptional responses.
21
Lam, Ka-man Amy. "Osmotic response element binding protein (OREBP) is an essential regulator of urine concentrating mechanism and renal protection." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B3127402X.
22
Berry, Andrew Edward. "Towards a molecular mechanism for light induction of gene transcription in Myxococcus xanthus." Thesis, University of Warwick, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340067.
23
Das, Sadhan Chandra. "Molecular mechanism of nucleolin-mediated Pol I transcription and characterization of nucleolin acetylation." Thesis, Lyon, École normale supérieure, 2012. http://www.theses.fr/2012ENSL0767.
Abstract:
Nous montrons dans cette étude que dans les cellules déplétées pour la nucléoline, une plus faible accumulation de pré-ARNr est associée à une augmentation de marques d’hétérochromatine (H3K9me2) et une diminution de marques d’euchromatine (H4K12ac et H3K4me3) sur la chromatine des gènes ribosomiques. Des expériences de ChIP-seq montrent que la nucléoline est enrichie dans la région codante et promotrice de l’ADNr et est préférentiellement associée avec les gènes non méthylés des ARNr. La déplétion de la nucléoline entraîne une accumulation de l’ARN Pol I au début de l’ADNr et une diminution de UBF sur la région codante et promotrice. La nucléoline interfère avec la liaison de TTF-1 sur le promoteur-proximal T0, inhibant ainsi le recrutement de TIP5 du complexe NoRC, et établissant un état d’hétérochromatine répressive. Ces résultats révèlent l’importance de la nucléoline dans le maintien d’un état euchromatinien des ADNr et dans l’élongation de la transcription. Nous montrons aussi dans cette thèse que l’acétylation est une nouvelle modification post-traductionnelle de la nucléoline. Des études d’immunofluorescence utilisant l’anticorps anti nucléoline acétylée montrent que la nucléoline acétylée est exclue des nucléoles. De plus, par ChIP-seq nous n’avons jamais pu détecter d’association significative de la nucléoline acétylée sur la chromatine des ADNr. Aussi, nous n’avons détecté aucune activation de la transcription de Pol II sur des matrices de chromatine avec la nucléoline acétylée. Nous trouvons une distribution de la nucléoline acétylée majoritairement dans le nucléoplasme où elle co-localise parfaitement avec le facteur d’épissage SC35, et partiellement avec les structures marquées avec un anticorps dirigé contre Y12, mais ne co-localise pas avec des structures contenant la coïline, ce qui suggère que cette fraction de la nucléoline pourrait être impliquée dans la synthèse ou le métabolisme des pré-ARNmHere we have shown that, in nucleolin depleted cells, lower accumulation of pre-rRNA is associated with the increase in heterochromatin marks (H3K9me2) and decrease of the euchromatin histone marks (H4K12Ac and H3K4me3) in rDNA chromatin. ChIP-seq experiments show that nucleolin is enriched in the coding and promoter region of the rDNA and is preferentially associated with the unmethylated rRNA genes. Nucleolin knockdown results in the accumulation of RNAPI at the beginning of the rDNA and a decrease of UBF in the coding and promoter regions. Nucleolin is able to interfere with the binding of TTF-1 on the promoter-proximal terminator T0 thus inhibiting the recruitment of the NoRC subunit TIP5 and HDAC1 and establishing a repressive heterochromatin state. These results reveal the importance of nucleolin in the maintenance of the euchromatin state of rDNA and transcription elongation.In this thesis we have also shown that acetylation is a novel post-translational modification of nucleolin. Immuno-fluorescence studies using anti-acetylated nucleolin antibody illustrated that acetylated nucleolin is excluded from nucleoli and interestingly, neither could we detect any significant binding of ac-nucleolin on rDNA chromatin by doing ChIP-Seq, nor did we detect any activation of Pol II transcription with ac-nucleolin from DNA and chromatin templates. Moreover, we found acetylated nucleolin had a predominant nucleoplasmic distribution where it associates with the splicing factor SC35 and partially with the structures labeled with Y12 antibody, but not with coilin containing structures
24
Kozlowski, Marek. "The molecular mechanism of PARP1 activation and its downstream roles in ALC1-regulated transcription." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-182038.
25
Mello, Jill Ann 1966. "Transcription and mismatch repair in the mechanism of action of the anticancer drug cisplatin." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/42993.
Abstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 1997.Vita.
Includes bibliographical references (leaves 188-212).
cis-Diamminedichloroplatinum(II) (cis-DDP or cisplatin) is a powerful cytotoxin and anticancer therapeutic, used most effectively in the treatment of testicular and ovarian cancers. By contrast, the geometric isomer of cisplatin, trans- DDP, is comparatively non-toxic and fails to show significant antitumor activity. Cisplatin is believed to derive its cytotoxic effects from processes triggered by its reaction with DNA. The formation of cisplatin adducts can elicit many cellular responses, including inhibition of both DNA replication and transcription. Cisplatin DNA adducts are also specifically recognized by various proteins within the cell, and such cisplatin-damage recognition proteins have been previously suggested to play a role in the clinical efficacy of the drug. To date, however, the precise mechanism by which cisplatin lesions mediate the cytotoxic and antitumor activities of cisplatin remains elusive. The work in this dissertation evaluated two possible mechanisms by which cisplatin might exert its cytotoxic effects that had been heretofore largely unexplored. The first aspect of this work evaluated a model wherein the differential cytotoxic and antitumor activities of cisplatin and trans-DDP may result from a greater ability of cisplatin DNA damage to inhibit RNA transcription. A nonreplicating plasmid harboring the [beta]-galactosidase ([beta]-gal) reporter gene was modified in vitro with either of the two platinum compounds and transfected into human or hamster cell lines. The use of cell lines both proficient and deficient in nucleotide excision repair allowed the examination of transcriptional bypass independent of excision repair for each platinum compound. A two to three fold higher level of transcription was observed in both cell lines from plasmids containing trans-DDP adducts as compared to plasmids modified by cis-DDP. This difference in transcriptional activity was not decreased in human and rodent nucleotide excision repair deficient cell lines, indicating that more efficient excision repair of the trans- DDP adducts was not the cause of its lower ability to block transcription. The possibility that trans-DDP lesions are preferentially bypassed by RNA polymerase was examined by monitoring the elongation of [beta]-gal mRNA on damaged templates in vivo. Nascent [beta]-gal mRNA transcripts were recovered from nucleotide excision repair deficient xeroderma pigmentosum A cells transfected with platinated plasmids, and the extent of RNA synthesis was measured by using ribonuclease protection. The results showed that four-fold more trans-DDP than cis-DDP adducts were required to inhibit transcription elongation by 63 %. RNA polymerase II translocated past a single, representative DNA adduct of cisplatin and trans-DDP in vivo with an efficiency of 0- 16% and 60-76%, respectively. These data support the view that inhibition of transcription may contribute to the greater cytotoxicity of cis-DDP compared with its trans isomer. The second aspect of this work evaluated a possible novel role of the human mismatch repair protein, hMSH2, as a cisplatin-damage recognition protein. The interaction of purified recombinant hMSH2 with DNA containing adducts of cisplatin and various cisplatin analogs was examined in vitro by using an electrophoretic gel mobility shift assay. The results showed that hMSH2 recognizes and binds specifically to DNA adducts of cisplatin. This protein displayed affinity for DNA modified by therapeutically effective platinum complexes, but not for that modified by clinically inactive platinum compounds such as trans-DDP. Recognition by hMSH2 was dictated, in part, by the major intrastrand DNA adduct formed by cisplatin. The results also show that hMSH2 is overexpressed in testicular and ovarian tissue, tissues in which tumors are best treated by cisplatin. These results complement a growing body of literature correlating mismatch repair activity with cisplatin toxicity in Escherichia coli and mammalian cells. Viewed together, these observations are consistent with a model whereby mismatch repair plays an active role in potentiating cisplatin DNA lesion toxicity. Further, these results may provide insight into a previously undiscovered mechanism by which tumor cells may acquire resistance to CISplatin.
by Jill Ann Mello.
Ph.D.
26
Laver, Travis. "Mechanism of inteferon-beta-mediated inhibition of IL-8 gene expression." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/laver.pdf.
27
Miyata, Kenji Sean. "The molecular mechanism of transcriptional activation by the peroxisome proliferator activated-receptor (alpha) /." *McMaster only, 1999.
28
Drane, Emma Louise Antoinette. "Mechanisms of human papillomavirus and host gene transcriptional deregulation in cervical carcinogenesis." Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/270255.
Abstract:
Cervical malignancy is the fourth most common cause of cancer-related mortality in women worldwide; infection with high-risk human papillomavirus (HRHPV) is responsible for over 500,000 cases of cervical carcinoma each year, approximately 90% of which are squamous cell carcinomas (SCCs). Over half of all HPV-positive cervical SCCs are caused by the deregulated expression of HPV16 oncogenes E6 and E7 in proliferating basal cells of the cervical squamous epithelium. The major risk factor associated with cervical neoplastic progression is integration of HRHPV into the host genome, which is detected in $~$85% of HPV16-positive cervical carcinomas. The work presented in this doctoral thesis sought to provide insights into our understanding of the process of HPV16 integration as well as to elucidate mechanisms that deregulate both virus and host gene expression following integration. The W12 cell model system used in this project is a polyclonal cervical keratinocyte line generated by explant culture of a low-grade cervical squamous intraepithelial lesion (LSIL) that arose following natural infection with HPV16. The W12 clones were isolated in the absence of selective pressure, and as such represent the range of integration events that occur in a pre-malignant lesion at the early stages of carcinogenesis, prior to integrant selection. Despite identical genetic backgrounds, expression levels of oncogenes E6 and E7 varied up to 16-fold between the W12 clones. Expression of HPV oncogenes is ultimately determined by transcription factor binding to the non-coding long control region (LCR) of the viral genome. The initial result of this study found that genomic mutations affecting transcription factor binding at the LCR of the W12 clones was not a cause of differential viral expression, concluding that epigenetic control may be at play. Higher levels of virus expression per template were associated with increased levels of histone post-translational modification (PTM) hallmarks of transcriptionally active chromatin and reduced levels of repressive hallmarks. There was greater abundance of the active/elongating form of the RNA polymerase-II enzyme (RNAPII-Ser2P), together with CDK9, the component of positive transcription elongation factor-b (P-TEFb) responsible for the Ser2 phosphorylation. The changes observed were functionally significant, as cells with higher HPV16 expression per template showed greater sensitivity to depletion and/or inhibition of histone acetyl transferases and CDK9, as well as reduced sensitivity to histone deacetylase inhibition. Employing next generation sequencing data available for five representative W12 clones, the sites of HPV16 host integration were identified. The three-dimensional (3D) structure of the nucleus and physical interactions between stretches of the genome over long distances (i.e. enhancer and promoters) are known to exert an additional level of gene regulation. Identification of 3D virus-host interactions in the W12 clones employing the newly developed and unique 'Sequence Capture of Regions Interacting with Bait Loci Hi-C' (SCRiBL-Hi-C) protocol showed that both short- ($~$50 kb), and long-range ($~$1 Mb) interactions occur during the early stages of carcinogenesis. Together, the data in this thesis indicate that transcription and subsequent expression of the HPV16 genome is controlled by multiple layers of epigenetic regulation.
29
Lam, Ka-man Amy, and 林嘉敏. "Osmotic response element binding protein (OREBP) is an essential regulator of urine concentrating mechanism and renal protection." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B3127402X.
30
Ali, Yousuf O. "The Mechanism of Neuroprotection Mediated By Nicotinamide Mononucleotide Adenylyl Transferase (NMNAT)." Scholarly Repository, 2011. http://scholarlyrepository.miami.edu/oa_dissertations/633.
Abstract:
Neurons need to be maintained to persist throughout adulthood for proper brain function. However neuronal activity, injury and aging exert physical stress on the nervous system, which compromise nervous system function. Healthy neurons are able to maintain their integrity throughout the lifespan of the animal, suggesting the existence of a maintenance mechanism that allows neurons to sustain or even repair damage. A forward genetic screening in Drosophila identified mutations in a gene called nmnat that cause a rapid and severe neurodegeneration immediately post neuronal differentiation and development. NMNAT protein was required to maintain neuronal integrity in an activity-dependent manner. When probing for the exact role of NMNAT in neuronal maintenance, a novel stress responsive chaperone function was identified, in addition to its essential housekeeping NAD synthase role. In this work, the mechanism of NMNAT-mediated neuroprotection is investigated. First, the transcriptional regulation of Drosophila NMNAT during acute stress is analyzed. Here, both stress transcription factors heat shock factor (HSF) and hypoxia inducible factor alpha (HIF1-α) have been shown to upregulate NMNAT during stress through a heat shock element in the nmnat promoter. In addition, the role of NMNAT for stress tolerance in Drosophila is revealed. Second, to elucidate the neuroprotective capacity of NMNAT in neurodegenerative disease, mouse models of tauopathy have been used. In the P301L Tau-transgenic mouse model, the levels of endogenous NMNAT2 have been studied at various ages to link a reduction in NMNAT2 as a precursor for neurodegeneration. The underlying mechanism of NMNAT2 downregulation is further studied in this model. Third, using Drosophila model of Tauopathy, the protective capacity of both wild type and enzyme-inactive NMNAT in ameliorating the pathological and behavioral impairments from Tau-induced neurodegeneration were studied extensively. The possible protective mechanism of NMNAT is uncovered by identifying novel interactions of NMNAT with hyperphosphorylated and ubiquitinated Tau in regulating the levels of toxic Tau species. Finally, this study also identified endogenous proteins that NMNAT interacts with to provide insight into a neuroprotective chaperone role of NMNAT. Together, these studies improve our understanding of the mechanisms of neuronal maintenance, by providing a comprehensive investigation of the stress-responsive regulation of NMNAT in both Drosophila and mammalian models, and its role as a chaperone both in protein foldopathies and in healthy neurons.
31
Margue, Christiane. "The anti-apoptotic function of paired box transcription factors : mechanism and importance for tumor biology /." [S.l.] : [s.n.], 2000. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=13765.
32
Zeng, Chunxi. "Riboswitch-targeted Drug Discovery: Investigation of Factors that Affect the T Box Transcription Antitermination Mechanism." Ohio University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1451943674.
33
Kumar, Deepak. "Mechanism of induction of matrix metalloproteinase-1 (MMP-1) during osteoarthritis /." Free to MU Campus, others may purchase, 2004. http://wwwlib.umi.com/cr/mo/fullcit?p3144432.
34
Wang, Zhibin. "Molecular mechanism of Arabidopsis CBF mediated plant cold-regulated gene transcriptional activation." Columbus, Ohio : Ohio State University, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1158600906.
35
Voleti, Bhavya. "Mechanism of Transcriptional Regulation of C-Reactive Protein Gene Expression." Digital Commons @ East Tennessee State University, 2007. https://dc.etsu.edu/etd/2058.
Abstract:
C-reactive protein (CRP) is an acute phase protein produced by hepatocytes whose serum concentration increases in inflammatory conditions including cardiovascular complications. Statins that are used in the treatment of cardiovascular diseases to reduce cholesterol also lower serum CRP levels. In human hepatoma Hep3B cells, CRP is induced in response to cytokines IL-6 and IL-1β. The objective of the study was to determine the mechanism of regulation of CRP gene expression in Hep3B cells in response to cytokines and to determine the effect of statins on CRP expression. Key findings of our research were: 1. IL-1β-activated NF-κB p50/p65 acted synergistically with IL-6-activated C/EBPβ in inducing CRP transactivation through the proximal CRP promoter. 2. A NF-κB site was localized in the proximal CRP promoter centered at position -69 overlapping the known OCT-1/HNF-1/HNF-3 sites. 3. The synergy between IL-6 and IL-1β in inducing CRP gene expression was partially mediated through the NF-κB site. 4. In the absence of C/EBPβ, a complex containing C/EBPζ and RBP-Jκ was formed at the C/EBP-p50-site. 5. Overexpressed C/EBPζ repressed both (IL-6+IL-1β)-induced and C/EBPβ-induced CRP expression. 6. OCT-1 repressed (IL-6+IL-1β)-induced CRP transactivation through the proximal CRP promoter. 7. Statins reduce cytokine-induced CRP gene expression at the transcriptional level. These findings led us to conclude that: 1. CRP transcription is determined by the relative levels of various transcription factors such as C/EBPβ, C/EBPζ, NF-κB and OCT-1 and their interaction with the proximal CRP promoter. 2. Inhibition of CRP transcription by statins is not due to an anti-inflammatory effect but due to the direct effect on CRP gene expression.
36
Sevostiyanova, Anastasia K. "Mechanism of Antitermination by NusG-like Proteins and the Role of RNAP Conformational Mobility in Transcription Cycle." The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1281629313.
37
Su, Kathy. "Mechanism of regulatory evolution : studies on the gain and diversification of wing pigmentation patterns in Drosphila." Thesis, Aix-Marseille 2, 2011. http://www.theses.fr/2011AIX22060/document.
Abstract:
La génération de la diversité morphologique n’implique pas seulement l’émergence de nouveaux caractères morphologiques, mais aussi la modification de caractères pre-existants. Des changements dans l'expression génique sous-tendent l’apparition et la divergence de morphologies au cours de l'évolution. Les variations de l'expression d'un gène peuvent résulter des modifications dans la séquence nucléotidique de ses éléments cis-régulateurs (ECRs) et/ou dans la séquence codante des facteurs de transcription (FTs) qui le régulent. En conséquence, la compréhension de l'évolution d'un nouveau trait exige l'élucidation des modifications en cis et en trans expliquant les différences d'expression des gènes. Les études sur la pigmentation des drosophiles ont considérablement contribué à notre compréhension du rôle joué par la modification des ECRs dans la diversification morphologique. Pour comprendre les mécanismes génétiques et moléculaires qui sous-tendent l’émergence et la diversification des traits morphologiques, nous avons décidé d’étudier l'évolution des motifs de pigmentation de l’aile chez les mouches du groupe d'espèces Drosophila melanogaster. Nous constatons que la diversification des modes de pigmentation de l’aile dans ce groupe est en corrélation avec la divergence d'expression d'un gène de pigmentation, yellow. Nous avons choisi de disséquer la régulation transcriptionnelle de yellow dans l’une des espèces tachetées, Drosophila biarmipes, avec pour objectif d'élucider les mécanismes responsables de la diversification de l'expression de yellow.Drosophila biarmipes, une espèce proche de Drosophila melanogaster, a développé une nouvelle tache de pigmentation sur aile qui est absente chez D. melanogaster. L'évolution de ce caractère est corrélée au changement d’expression de yellow. Une précédente étude a identifié, dans la région 5' du promoteur de yellow de D. biarmipes, un ECR qui suffit à reproduire une partie de l'expression de yellow lorsqu’il est transformé chez D. melanogaster. Avec un crible RNAi, j’ai identifié cinq FTs qui peuvent réguler l’ECR de yellow. Un de ces candidats, Distal-less, est exprimé d’une façon qui correspond à celle de l'expression de yellow et au profil de la pigmentation de l’aile chez D. biarmipes. Une analyse comparative de Distal-less et yellow a mis en évidence que les profils d'expression de ces gènes sont également corrélés avec les différents patrons de pigmentation dans les ailes d’autres espèces de drosophiles. J’ai aussi pu montrer que la surexpression de Distal-less dans l’aile de D. biarmipes est suffisante pour induire de la pigmentation ectopique. Ceci suggère que Distal-less ne régule pas seulement yellow, mais l’ensemble des gènes de pigmentation nécessaires à l’apparition de tache chez D. biarmipes. Mon travail montre que l'évolution de nouveaux motifs de pigmentation a commencé par le recrutement de Distal-less sur une batterie de gènes pigmentation. A partir de ces constatations, nous proposons un modèle expliquant les mécanismes de régulation conduisant à l'émergence et la diversification des motifs de pigmentation de l'aile au sein du groupe d'espèces melanogaster. Nous suggérons que dans un ancêtre commun de ce groupe, Distal-less a été recruté par une batterie des gènes de pigmentation, qui conduisent à l'émergence d'un motif de pigmentation. Par ailleurs, nous faisons l’hypothèse qu’une fois un lien régulatoire a été formé entre Distal-less et les gènes de pigmentation, alors la divergence d'expression de Distal-less pourrait entraîner des changements dans la régulation spatiale de tous les gènes de pigmentation donnant lieu à la diversification des motifs de pigmentationThe generation of morphological diversity involves not only the emergence of novel morphological traits but also the modification of pre-existing ones. To understand the genetic and molecular mechanisms underlying the gain and diversification of morphological traits, we chose to study the evolution of wing pigmentation patterns in a group of flies in the melanogaster species group. We find that the diversification of wing pigmentation patterns in this group is mirrored by the divergence of expression of a pigmentation gene, yellow. To decipher the regulatory mechanisms underlying the diversification of yellow expression, we chose to dissect the transcriptional regulation of yellow in one spotted species, D. biarmipes. A functional dissection of the yellow spot enhancer, situated at the 5’ of the D. biarmipes yellow promoter, shows that a homeobox transcription factor, Distal-less (Dll), is a direct activator of yellow. Moreover, we show that ectopic expression of Dll is sufficient to induce ectopic pigmentation in D. biarmipes, which suggests that Dll is regulating yellow and other pigmentation genes. Furthermore, we find that the divergence of Yellow expression pattern in the melanogaster species group is correlated with the divergence in expression of Dll. Based on our findings we propose a model to explain the regulatory mechanisms that lead to the emergence and diversification of wing pigmentation patterns within the melanogaster species group. We suggest that in a common ancestor of all spotted species within the Oriental lineage, there was a gain of regulatory links between Dll and terminal pigmentation genes, such as yellow, which lead to the emergence of an ancestral pigmentation pattern. We further propose that once a regulatory link was formed between Dll and terminal pigmentation genes, divergence of Dll expression could lead to changes in the spatial regulation of all downstream target genes, which would give rise to the diversification of pigmentation patterns. Our study illustrates how key regulators of terminal genes could be targets for mutations that are more likely to lead to morphological diversification
38
Tao, Jing. "PAK1-Nck Interaction Represents a Novel Mechanism to Regulate Cyclin D1 Transcription in Response to Prolactin." University of Toledo / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1279319314.
39
Buser, Adam C. "Mechanism of progesterone receptor repression of transcription of the [beta]-casein gene in mammary epithelial cells /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2007.
Abstract:
Thesis (Ph.D. in Cancer Biology) -- University of Colorado Denver, 2007.Typescript. Includes bibliographical references (leaves 182-210). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;
40
Ravanpay, Ali Cyrus. "Insights into the molecular interactions of the neurogenic basic helix-loop-helix transcription factor, neuroD2, and the mechanism of regulation of a key target, RE-1 silencing transcription factor /." Thesis, Connect to this title online; UW restricted, 2007. http://hdl.handle.net/1773/10628.
41
Wu, Qianchao [Verfasser], and Frank [Akademischer Betreuer] Lyko. "Regulation of p53 target gene transcription by a TBL1-mediated epigenetic mechanism / Qianchao Wu ; Betreuer: Frank Lyko." Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/119334736X/34.
42
Thowfeik, Fathima Shazna. "Targeting a Common Enemy: Toxic Cellular Mechanism of Novel Anti-cancer Agents that Alter DNA and Transcription." University of Cincinnati / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1460652655.
43
Khan, Md Gulam Musawwir. "Nuclear import mechanism of Php4 under iron deprivation in fission yeast Schizosaccharomyces pombe." Mémoire, Université de Sherbrooke, 2015. http://hdl.handle.net/11143/6008.
Abstract:
Php4 is a subunit of the CCAAT-binding protein complex that has a negative regulatory function during iron deprivation in the fission yeast Schizosaccharomyces pombe. Under low iron conditions, Php4 fosters the repression of genes encoding iron using proteins. In contrast, under iron-replete conditions, Php4 is inactivated at both transcriptional and post-transcriptional levels. Our group has already described that Php4 is a nucleo-cytoplasmic shuttling protein, which accumulates into the nucleus during iron deficiency. On the contrary, Php4 is exported from the nucleus to the cytoplasm in response to iron abundance. Php4 possesses a leucine-rich NES (93LLEQLEML100) that is necessary for its nuclear export by the exportin Crm1. Our current study aims at understanding the mechanism by which Php4 is imported in the nucleus during iron starvation. Through microscopic analyses using different mutant strains, we showed that the nuclear localization of Php4 is independent of the other subunits of the CCAAT-binding core complex namely Php2, Php3 and Php5. Deletion mapping analysis of Php4 identifies two putative nuclear localization sequences (NLSs) in Php4 (171KRIR174 and 234KSVKRVR240). Using chimeric proteins that consist of GFP fused to Php4, we engineered substitutions of the basic amino acid residues 171AAIA174 and 234ASVAAAA240 and analyzed the functionality of both NLSs. We observed that both monopartite NLSs play critical role for Php4 nuclear localization. We also observed that
mutant strains of cut15+, imp1+ or sal3+ exhibited defects in nuclear targeting of Php4, revealing that nuclear accumulation of Php4 is dependent on two karyopherin α (Imp1 and Cut15) and one karyopherin β (Sal3) receptors. Consistently, the Php4-mediated repression activity is abolished in the absence of two functional NLSs. Moreover, loss of Imp1, Cut15 or Sal3 resulted in increased expression of isa1+, which is a target gene of Php4. Co-immunoprecipitation assay (Co-IP) reveals physical interaction of Php4 with Imp1, Cut15 and Sal3 in vitro. Collectively, our results demonstrate that Php4 has two distinct NLS regions responsible for its nuclear localization. Furthermore, karyopherin α and β receptors play a role in the nuclear import of Php4. Because Php4 is essential for growth under low iron conditions, the presence of two NLSs would ensure the protein to reach its nuclear destination when cells undergo a transition from iron-sufficient to iron-limiting conditions.
44
Wissink, Sacha. "A mechanism for the anti-inflammatory action of glucocorticoids : repression of transcription factor NF-kappaB activity = Een mechanisme voor de anti-inflammatoire werking van glucocorticoïden /." [S.l. : s.n.], 1997. http://www.gbv.de/dms/bs/toc/254083013.pdf.
45
Nishida, Nao. "Studies on the mechanism of organic solvent tolerance of yeast Saccharomyces cerevisiae triggered by a transcription factor Pdr1p." Master's thesis, Kyoto University, 2014. http://hdl.handle.net/2433/188764.
Abstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第18326号
農博第2051号
新制||農||1022(附属図書館)
学位論文||H26||N4833(農学部図書室)
31184
京都大学大学院農学研究科応用生命科学専攻
(主査)教授 植田 充美, 教授 喜多 恵子, 教授 栗原 達夫
学位規則第4条第1項該当
46
Whiting, Sam H. "Studies into the characteristics and mechanism of strand displacement synthesis by retroviral reverse transcriptase /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/11494.
47
Kozłowski, Marek [Verfasser], and Andreas [Akademischer Betreuer] Ladurner. "The molecular mechanism of PARP1 activation and its downstream roles in ALC1-regulated transcription / Marek Kozłowski. Betreuer: Andreas Ladurner." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2015. http://d-nb.info/1072376334/34.
48
Ishii, Tetsu. "Molecular mechanism of activation of transcription factor NF-kB by the mutants of the interferon-inducible protein kinase PKR." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=33782.
Abstract:
The i&barbelow;nterf&barbelow;eron&barbelow; (IFN) inducible d&barbelow;ouble s&barbelow;tranded (ds) RNA activated protein kinase PKR inhibits protein synthesis by phosphorylating the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF-2alpha). The introduction of dominant negative mutants of PKR leads to cell transformation and tumor formation in nude mice, and is thought to be due to the deregulation of general protein synthesis. In addition to translational control, PKR has been implicated in several signaling pathways leading to gene transcription. For example, a link between PKR and NF-kappaB activation was first detected when PKR knockout mouse embryonic fibroblasts were deficient in NF-kappaB DNA binding upon dsRNA mediated activation. Recently, PKR has been shown to induce IkappaB kinase (IKK) activity resulting in the induction of NF-kappaB mediated gene transcription. The mechanism of activation of IKK by PKR has been suggested to be independent of PKR's catalytic capacity and may be due to a protein-protein interaction between PKR and IKK. The effects of dominant negative mutants of PKR (PKRDelta6/PKRLS4/PKRLS9) on the activation of NF-kappaB was examined in NIH3T3 cells. (Abstract shortened by UMI.)
49
Lerche, Michael. "Elucidating the activation mechanism of the transcription factor DntR using X-ray crystallography and small angle X- ray scattering." Thesis, Grenoble, 2014. http://www.theses.fr/2014GRENV013/document.
Abstract:
Les protéines régulatrices de la transcription de type LysR (LTTR) appartient à la plus grande famille de facteur de transcription chez les procaryotes. Malgré l'importance de cette famille, les informations structurelles sur les protéines pleine-longueur sont très limitées car elles sont souvent insolubles et très difficiles à cristalliser. Les quelques structures existantes, couplés à d'autres analyses biophysiques ont pu montrer que ces protéines s'associent principalement sous forme d'homotétramère comprenant un dimère de dimères. Les dimères s'associent par un large domaine C-terminal dans une position " tête-bêche " et sont reliés en " tête-à-tête " par leurs domaines N-terminal et sont activées par la liaison de molécules inductrices. Le domaine dimèrique C-terminal qui contient la poche de liaison inductrice (Inducer Binding Cavity : IBC) est appelé domaine de liaison inductrice (Inducer Binding Domain : IBD), tandis que les dimères N-terminaux se lient chacun à une région de l'ADN par un motif hélice-tour-hélice ‘winged' (wHTH). Contrairement à d'autres facteurs de transcription, les protéines LTTR ne régulent pas l'expression par association/dissociation avec l'ADN. Ils se lient à l'ADN dans leur état actif et inactif. Le consensus actuel est qu'elles régulent l'expression des gènes par d'importants changements conformationnels qui relâchent la liaison avec l'ADN. À ce jour, aucune structure de LTTR pleine longueur homotétramérique dans une conformation active ou inactive n'a été résolu par cristallographie, et leur mécanisme d'action sur le gène reste structurellement non caractérisé.Le travail décrit dans cette thèse a utilisé DntR de la famille des LTTR. La première structure cristalline de l'apo-DntRis est présentée ici, ainsi que la structure du mutant H169TDntR, qui présente une activité en l'absence d'inducteur. L'analyse par fluorimétrie de différentiel thermique (TSA) montre que la température de dénaturation du mutant H169TDntR est similaire à DntR IBDs lié à une molécule inductrice. La comparaison de ces deux structures avec celle de DntR lié au salicylate révèle que la protéine dans son état apo adopte une conformation compacte de l'IBC, ce qui empêche la liaison d'une molécule inductrice. Dans l'IBC, les mouvements des résidus H169 et H206 permettent la liaison à l'inducteur. Pour éviter les limitations dues à l'empaquetage du cristal nous avons étudié la structure DntR en solution par diffusion des rayons X aux petits angles (SAXS).L'étude SAXS de DntR révèle que dans son état inactif, la conformation apo adopte un repliement plus compact par rapport à celle de la structure cristalline. Tout en maintenant un noyau compact de C-terminal, le repliement du dimère de wHTH est beaucoup plus fermé que dans la structure cristalline et adopte une conformation qui entrainerait une flexion beaucoup plus importante de l'ADN lié que postulé précédemment. Les études du mutant H169TDntR constitué actif ont confirmé comme l'analyse par TSA l'a suggéré que, la structure de cette protéine est nettement différente en solution que sous forme cristalline.En effet, la structure en solution de H169TDntR est très semblable à la forme ouverte de l'homotétramères observés dans la structure cristalline de TsaR. L'hypothèse de départ était que, lors de l'activation de LTTR, cet homotétramère subirait un changement de conformation d'une forme compact vers une forme ouverte, qui se traduirait par un relâchement de l'ADN lié. Cette hypothèse a été confirmée par des études de diffusion en solution de DntR activée par un inducteur.Le travail présenté dans cette thèse valide l'hypothèse précédemment, que lors de l'activation de DntR, et probablement tous les LTTRs homotétramériques, entraine un changement de conformation d'une forme compacte vers une forme beaucoup plus ouverte et permet l'accès aux régions promotrices par l'ARN polymerase et ainsi initier la transcriptionLysR type transcriptional regulatory (LTTR) proteins are the largest family of transcription factors amongst prokaryotes. In spite of the size of the family, structural information on full-length constructs of these proteins is very limited as they are often insoluble and very difficult to crystallize. From the few existing crystal structures, coupled with other biophysical evidence, it is known that the proteins mainly associate as homotetramers comprising a dimer of dimers. The dimers associate through large C-terminal domains in a “head-to-tail” fashion and are connected “head-to-head” through their N-terminal domains and the resulting homotetramers are activated by the binding of inducer molecules. Each C-terminal domain contain an inducer binding cavity (IBC) and is denoted an inducer binding domain (IBD), while the N-terminal dimers each bind a region of DNA via a winged helix-turn-helix (wHTH) motif.Unlike other transcription factors, LTTR proteins do not regulate expression by associating or disassociating with DNA. They bind to DNA in both their active and inactive states and the current consensus is that they regulate gene expression through large conformational changes that relax the bending of bound DNA. However, to this date, no crystal structures of a full length homotetrameric LTTR in both an active and inactive conformation exists, and thus their mechanism of transcriptional regulation remains structurally uncharacterized.The work described in this thesis has used the LTTR DntR as a model protein to futher structurally characterizes the activation mechanism of LTTR proteins. The first crystal structure of apo-DntR is presented as is the crystal structure of H169TDntR, a mutant which shows activity in the absence of an inducer molecule. Thermofluor assays performed on this mutant, show that it has a melting temperature similar to that of inducer bound DntR. Comparison of these crystal structures with the crystal structure of salicylate-bound DntR reveals that the protein in its apo-state adopts a compact IBC, which precludes the binding of an inducer molecule. Despite the evidence of thermofluor assays, the crystal structure of H169TDntR is very similar to that of apo-DntR suggesting that crystal packing effects impose strong limitations on the use of crystallography to elucidate the active and inactive conformations of DntR. Small Angle X-ray Scattering (SAXS) was thus used to study the structure of DntR in solution.SAXS study reveals that in solution DntR in its inactive apo-state is found in a slightly different conformation compared to that seen in its crystal structure. While maintaining a compact tetrameric C-terminal core the DNA binding wHTH dimers pack much closer to this than seen in the crystal structure and adopt a conformation that would result in much higher bending of bound DNA than previously postulated.SAXS studies of the constitutively active H169TDntR mutant confirm, as thermofluor assays had suggested, that in solution the structure of this protein is markedly different from its crystal structure. Indeed the solution structure of H169TDntR appears very like that of open-form homotetramers seen in the crystal structure of TsaR. This same effect was observed in solution scattering studies of inducer bound-and thus activated, DntR.The work presented in this thesis thus appears to confirm, as previously hypothesized, that upon activation DntR, and presumably all homotetrameric LTTRs, undergo a conformational change from a compact, to a much more open form that allows the relaxation of the bound DNA promoter region, exposing it to solvent and allows RNA polymerase access and thus initiate transcription
50
Anderson, Jeffrey A. "Retroviral recombination during reverse transcription an analysis of the mechanism, frequency, and effect of the viral packaging signal [psi] /." Morgantown, W. Va. : [West Virginia University Libraries], 2001. http://etd.wvu.edu/templates/showETD.cfm?recnum=1822.
Abstract:
Thesis (Ph. D.)--West Virginia University, 2001.Title from document title page. Document formatted into pages; contains viii, 174 p. : ill. Vita. Includes abstract. Includes bibliographical references.