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dc.contributor.advisorShearwin, Keithen
dc.contributor.advisorDodd, Ianen
dc.contributor.authorCui, Lunen
dc.description.abstractProtein binding to DNA sequences is the foundation of transcriptional regulation. By binding at specific DNA sequences, such as promoters, proteins can recruit other proteins to regulate gene transcription. Some proteins, which bind at separated DNA binding sites, can interact via the formation of a DNA loop. DNA looping is essential in many processes, such as replication, recombination and gene regulation. In prokaryotic systems, DNA looping is involved in some genetic switches, which can control bacteriophage lysogenic/lytic pathways or bacterial catabolic pathways. Small DNA loops are essential for effective repression of several operons in bacteria. In eukaryotic systems, enhancers are distal gene regulatory elements, which can be located far away from the promoter. The formation of DNA looping is thought to be necessary for the function of enhancers. Bacteriophage λ CI repressor activates the transcription of its own gene, while the CI mediated looping represses its own transcription. This DNA looping mediated long-range repression improves the efficiency of the lambda lysogenic/lytic switch. However, evidence in the literature suggests an additional activation effect of λ CI DNA looping. In Chapters 2 and 3, I investigated this long-range λ CI DNA looping mediated transcriptional activation. By using a synthetic λ CI DNA looping reporter system, I confirmed that λ CI DNA looping can mediate enhancer-like long-range transcriptional activation. In vivo experiments showed that the λ PRM promoter was activated by the α C-terminal domain (CTD) of RNA polymerase contacting an UP element located 2.3 kilobases away from the PRM promoter. A physicochemical model of the in vivo data showed that an RNA polymerase α subunit recruitment mechanism could fully explain this activation effect. DNA-protein structural modelling found that the bending of linker sequence between OL2 and the UP element is required for the contact. The efficiency of long range DNA looping has been studied in Chapter 4. In vivo Lac looping and lambda CI DNA looping constructs were used to generate data for calculating DNA looping efficiency. DNA loop sizes ranging from 250 bp to 10000 bp were tested. Tethered particle motion (TPM) experiments, performed by our collaborators, generated in vitro DNA looping data by using Lac mediated DNA loops ranging from 600 bp to 3200 bp. Based on these in vitro and in vitro data, mathematical modelling calculated DNA looping parameters for understanding DNA looping efficiency. The insertion of DNA looping constructs into the E.coli chromosome (by using a bacteriophage integrase based approach) also led us to make a one-step integration system (OSIP), described in Chapter 5. The OSIP system is a set of OSIP plasmids, which can mediate one-step bacterial chromosomal integration of DNA sequences. The cloning module of each OSIP plasmid has both the integrase gene and corresponding att sequences, which are required for integrating the OSIP plasmid into the bacterial chromosome. An integration protocol, called clonetegration, was developed by coupling the OSIP system with in vitro isothermal DNA assembly. Clonetegration bypasses plasmid propagation and purification procedures by transferring assembly products directly into target competent cells.en
dc.subjectgene regulation; DNA looping; synthetic biology; genome editingen
dc.titleDNA looping mediated transcriptional regulation.en
dc.contributor.schoolSchool of Molecular and Biomedical Scienceen
dc.provenanceThis electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
dc.provenanceCopyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.en
dc.description.dissertationThesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2014en
Appears in Collections:Research Theses

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