Interdomain Repression in the Enhancer Binding Protein NorR

Bush, Matthew (2011) Interdomain Repression in the Enhancer Binding Protein NorR. Doctoral thesis, University of East Anglia.

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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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2259 not found.
Date Deposited: 07 Mar 2014 09:22
Last Modified: 07 Mar 2014 09:22


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