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Functional analysis of the DNA-binding domain of the Staphylococcus aureus master virulence transcription factor AgrA

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Title: Functional analysis of the DNA-binding domain of the Staphylococcus aureus master virulence transcription factor AgrA
Authors: Nicod, Sophie
Item Type: Thesis or dissertation
Abstract: The LytTR domain is a DNA-binding domain that is present in 5% of all DNA-binding transcription factors (TFs). The LytTR family of DNA-binding TFs is poorly characterised but crucially important as LytTR domain-containing TFs (LDCTFs) are disproportionately associated with virulence genes regulation. Although some members of the family have been extensively studied, how LDCTFs activate transcription remains to be determined. The accessory gene regulator A (AgrA) in Staphylococcus aureus (S. aureus) is the master TF responsible for the activation of transcription at the agr operon, a key regulatory locus involved in the switch between early colonisation and the spread of infection. AgrA is an LDCTF and the only one for which the crystal structure of the LytTR domain in complex with DNA is available. Here an experimental system was developed to study AgrA activity in vivo in order to try to unravel the mechanism of transcription activation by LDCTFs using AgrA as a model. A systematic alanine scanning mutagenesis of the LytTR domain of AgrA was performed and the mutants were characterised in vivo and in vitro. Overall, the results identify amino acid (aa) residues in the LytTR domain of AgrA that are important for DNA-binding and transcription activation and provide novel molecular and mechanistic insights on how AgrA and other LDCTFs activate transcription. More specifically, a key residue for transcription activation by AgrA, Y229, was identified. This residue is important for transcription activation but is not involved in the ability to bend DNA. These findings challenge the current assumption that the DNA bending activity of AgrA is responsible for its ability to activate transcription. We therefore propose a new model for transcription activation by AgrA based on our findings.
Content Version: Open Access
Issue Date: Sep-2014
Date Awarded: Jun-2015
URI: http://hdl.handle.net/10044/1/33244
DOI: https://doi.org/10.25560/33244
Supervisor: Wigneshweraraj, Sivaramesh
Tang, Christoph
Sponsor/Funder: Biotechnology and Biological Sciences Research Council (Great Britain)
Department: Department of Medicine
Publisher: Imperial College London
Qualification Level: Doctoral
Qualification Name: Doctor of Philosophy (PhD)
Appears in Collections:Medicine PhD theses

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