Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.543183
Title: Interdomain repression in the enhancer binding protein NorR
Author: Bush, Matthew
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2011
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Abstract:
NO (nitric oxide) is an intermediate of respiratory denitrification and is one of the toxic species released by macrophages of the immune system in the defence against invading pathogenic bacteria. In Escherichia coli, the expression of the Nitric Oxide (NO) reductase (NorVW) is tightly regulated by NorR, a member of the bacterial Enhancer Binding Protein (bEBP)-family that activates σ54-dependent transcription of the norVW genes under conditions of nitrosative stress. Binding of NorR to three conserved enhancer sites upstream of the norVW promoter is essential for transcriptional activation and promotes the formation of a stable higher-order nucleoprotein complex. NorR falls into a class of bEBPs that are negatively regulated – the regulatory (GAF) domain represses the activity of the ATPase (AAA+) domain in the absence of NO. NO binds to the non-heme iron centre of the GAF domain, stimulating ATP hydrolysis by the AAA+ domain and establishing an interaction between the activator and σ54 that leads to the remodelling of the closed promoter complex. However, the route by which NorR couples signal sensing to substrate remodelling is unknown. Here, the mechanism of interdomain repression in NorR has been investigated by characterising substitutions in the AAA+ domain that bypass repression by the regulatory domain. Most of these substitutions are located in the vicinity of the surface-exposed loops that engage σ54 during the ATP hydrolysis cycle or in the highly conserved GAFTGA motif that directly contacts σ54. A combination of genetic and biochemical approaches were used to show that the regulatory domain of NorR is unlikely to control AAA+ activity using previously characterised mechanisms, employed by related bEBPs. Instead, this work identifies a novel mechanism in which the σ54-interaction surface of the AAA+ domain is a target of the GAF-mediated repression mechanism. This hypothesis is further supported by EMreconstructions of two characterised escape-variants in their on-states, one of which represents the first structure of a bEBP bound to enhancer DNA. In the case of NorR, regulation at the point of σ54-interaction may be linked to the pre-assembly of an inactive hexamer, “poised” at the enhancer sites, enabling the cell to rapidly respond to nitrosative stress.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.543183  DOI: Not available
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