Issue 11, 2011
From the journal:

Molecular BioSystems

Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study

M. Ritzefeld,a   K. Wollschläger,†a   G. Niemann,a   D. Anselmettib  and  N. Sewald*a  

* Corresponding authors

a Organic and Bioorganic Chemistry, Bielefeld University, PO Box 100131, 33501 Bielefeld, Germany
E-mail: [email protected]
Fax: +49 521 106 8094
Tel: +49 521 106 2051

b Single Molecule BioPhysics & Systems NanoBiology, Bielefeld University, Bielefeld, Germany

Abstract

The two-component regulatory system PhoR/PhoB induces the expression of several genes in response to phosphate starvation in Escherichia coli. In order to quantify these protein–DNA interactions and to study the time-resolved dynamics of the binding mechanism, the specific recognition of different oligonucleotide duplexes by the DNA-binding domain of PhoB (PhoBDBD) was analyzed using surface plasmon resonance. In addition the two point mutants PhoBDBDD196A and PhoBDBDR219A were obtained and the DNA recognition in comparison to the wildtype PhoBDBD was investigated. Aspartic acid 196 and arginine 219 mediate specific minor groove interactions. All results reveal that at high PhoBDBD-concentrations all recognition sequences of the pho box are occupied. Decreasing the protein amount results in a mixture of free oligonucleotides and DNA molecules occupied by two WT-PhoBDBD. Moreover, the SPR results indicate that both binding site segments, the TGTCA-motif and the A/T-rich minor groove, are essential for the binding process. A comparison of different regulons additionally proved the dependency of the recognition process on the base composition of the minor groove.

Graphical abstract: Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study

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Article information

DOI
https://doi.org/10.1039/C1MB05281K
Article type
Paper
Submitted
05 Jul 2011
Accepted
19 Aug 2011
First published
12 Sep 2011

Mol. BioSyst., 2011,7, 3132-3142

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Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study

M. Ritzefeld, K. Wollschläger, G. Niemann, D. Anselmetti and N. Sewald, Mol. BioSyst., 2011, 7, 3132 DOI: 10.1039/C1MB05281K

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