Please use this identifier to cite or link to this item: http://hdl.handle.net/2440/99568
Type: Theses
Title: Testing the DNA loop domain model in Escherichia coli
Author: Priest, David Geoffrey
Issue Date: 2014
School/Discipline: School of Molecular and Biomedical Science
Abstract: The ability of DNA to form loops has been employed by evolution in almost every aspect of biology involving DNA, not least the regulation of gene transcription. The biophysical constraints on looping of the DNA polymer at short range (< 300 bp) have been extensively studied, however it is uncertain how the probability of DNA looping decays at longer range. The first part of this thesis presents a quantitative investigation of long range DNA looping both in vivo in E. coli and in vitro. DNA looping is more efficient in vivo than measured in vitro (by our collaborators) with the technique of Tethered Particle Motion (TPM), and we suggest that DNA supercoiling aids DNA looping in vivo. By measuring long-range looping in vivo using the two well-characterised looping proteins (the LacI and λCI repressors) and thermodynamic models of DNA looping, the decay in looping probability is quantified over the range 242–10000 bp. Furthermore this decay is shown to be a property of the DNA tether linking the loop, independent of the nature of the DNA looping protein(s). Enhancers activate genes at long distance irrespective of position and orientation, so why don’t enhancers activate the wrong genes? In other words, what mechanisms drive efficiency and specificity in enhancer-promoter looping? The loop domain model proposes that DNA loops formed by insulators pose a topological barrier that restricts the reach of enhancers to the vicinity of desired target promoter(s). Specifically, the model predicts that two DNA loops in an alternating arrangement should form somewhat mutually exclusively (i.e. they should interfere with one another’s formation), whereas nested DNA loops are predicted to assist one another’s formation, and side-by-side loops should form independently. In the second part of this thesis, the loop domain model is tested in E. coli by combining LacI and λCI-mediated DNA loops in these different orientations. Accordingly, we quantify DNA looping assistance and interference by fitting experimental data to a statistical-mechanical model, confirming the predictions of the loop domain model. Furthermore, TPM measurements of the same looping constructs support predictions that non-supercoiled DNA in vitro should facilitate DNA looping assistance, but not interference. In addition to confirming the loop domain model in E. coli, this thesis provides a strong experimental and theoretical foundation for further investigations of enhancer-promoter looping in prokaryotes and eukaryotes, and the relationship between chromatin architecture and gene expression in metazoans.
Advisor: Shearwin, Keith
Dodd, Ian
Dissertation Note: Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Molecular and Biomedical Science, 2014.
Keywords: E. coli
DNA looping
lac repressor
lambda repressor
loop domain model
genetics
biochemistry
Provenance: This 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: http://www.adelaide.edu.au/legals
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