Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.

Détails

ID Serval
serval:BIB_D3E453C01AFA
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
Characterization of HbpR binding by site-directed mutagenesis of its DNA-binding site and by deletion of the effector domain.
Périodique
FEBS Journal
Auteur(s)
Tropel D., van der Meer J.R.
ISSN
1742-464X[print], 1742-464X[linking]
Statut éditorial
Publié
Date de publication
2005
Volume
272
Numéro
7
Pages
1756-1766
Langue
anglais
Résumé
In the presence of 2-hydroxybiphenyl, the enhancer binding protein, HbpR, activates the sigma54-dependent P(hbpC) promoter and controls the initial steps of 2-hydroxybiphenyl degradation in Pseudomonas azelaica. In the activation process, an oligomeric HbpR complex of unknown subunit composition binds to an operator region containing two imperfect palindromic sequences. Here, the HbpR-DNA binding interactions were investigated by site-directed mutagenesis of the operator region and by DNA-binding assays using purified HbpR. Mutations that disrupted the twofold symmetry in the palindromes did not affect the binding affinity of HbpR, but various mutations along a 60 bp region, and also outside the direct palindromic sequences, decreased the binding affinity. Footprints of HbpR on mutant operator fragments showed that a partial loss of binding contacts occurs, suggesting that the binding of one HbpR 'protomer' in the oligomeric complex is impaired whilst leaving the other contacts intact. An HbpR variant, devoid of its N-terminal sensing A-domain, was unable to activate transcription from the hbpC promoter while maintaining protection of the operator DNA in footprints. Wild-type HbpR was unable to activate transcription from the hbpC promoter when delta A-HbpR was expressed in the same cell, suggesting the formation of (repressing) hetero-oligomers. This model implies that HbpR can self-associate on its operator DNA without effector recognition or ATP binding. Furthermore, our findings suggest that the N-terminal sensing domain of HbpR is needed to activate the central ATPase domain rather than to repress a constitutively active C domain, as is the case for the related regulatory protein XylR.
Mots-clé
Bacterial Proteins/chemistry, Bacterial Proteins/metabolism, Base Sequence, Binding Sites, DNA Footprinting, DNA-Binding Proteins/chemistry, DNA-Binding Proteins/metabolism, Electrophoretic Mobility Shift Assay, Molecular Sequence Data, Mutagenesis, Site-Directed, Mutation, Promoter Regions, Genetic, Pseudomonas/genetics, Pseudomonas/metabolism, Trans-Activators/chemistry, Trans-Activators/metabolism
Pubmed
Web of science
Open Access
Oui
Création de la notice
21/01/2008 13:36
Dernière modification de la notice
20/08/2019 15:53
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