Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.716444
Title: Studies of iron-sulfur cluster containing regulators of the Rrf2 family
Author: Pellicer Martinez, Maria
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2017
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Abstract:
Members of the Rrf2 superfamily of transcription factors are widespread in bacteria but their biological functions are largely unknown. The aim of the work described in this thesis was to uncover the distinct iron-sulfur cluster properties and biological functions of two uncharacterised members of the Rrf2 family. RirA (Rhizobial iron regularor A) is a global iron regulator in Rhizobium and many related α-proteobacteria. Spectroscopic and ESI-MS data for cluster-reconstituted RirA indicated that the protein binds a [4Fe-4S] cluster cofactor. [4Fe-4S] RirA binds DNA, consistent with its function as a repressor of expression of genes involved in iron uptake. Under low iron conditions, [4Fe-4S] RirA undergoes cluster conversion resulting in a [2Fe-2S] form, which binds DNA with lower affinity. If low iron conditions persist, the [2Fe-2S] cluster is lost, resulting in a cluster-free (apo) form. This does not bind DNA and can no longer function as a repressor and so the iron-uptake machinery of the cell is activated. The data are consistent with RirA functioning as a sensor of iron via both ironsulfur cluster availability and the fragility of its cluster. The significant sensitivity of RirA to O2 suggests that even when iron is sufficient, the protein is susceptible to cluster conversion/loss; the interplay between iron and O2 might be important in vivo. A new member of the Rrf2 superfamily from Streptomyces, RsrR (Redox sensitive response Regulator), was also characterised. RsrR is isolated containing a [2Fe-2S] cluster than undergoes facile redox cycling. This modulates its DNA-binding behaviour: it binds RsrR-regulated promoter DNA tightly only in its oxidised state. Spectroscopic analyses revealed that the cluster has properties characteristic of His-coordinated [2Fe-2S] clusters and His12 was identified through site-directed mutagenesis as a likely cluster ligand. Substitution of Glu8 also significantly affected the cluster properties suggesting it might also be a cluster ligand.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.716444  DOI: Not available
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