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Bielefeld University > Department of Chemistry > Organic Chemistry III > Team > Former

Personal data for Gesa Niemann

  Gesa Niemann
Gesa Niemann
PhD Student
Room: F2-219
Phone: 0521 106 2151
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DNA-protein interactions between transcription factor PhoB from E. coli and corresponding DNA sequences


Specific molecular recognition between DNA and proteins is essential for the regulation of fundamental biological processes, including DNA replication, repair and transcription.

The purpose of this study is to enhance the understanding of the interaction between DNA and DNA binding proteins. The DNA binding domain (DBD) of the transcription factor PhoB from E. coli is investigated as a model system. Structurally, PhoB belongs to the family of winged helix-turn-helix proteins and controls the expression of genes involved in phosphate metabolism [1]. It contains a transactivation domain and the DBD that binds to DNA sequences located within specific Pho-box DNA motifs.



The modified DBD of PhoB is generated by recombinant expression in E. coli and affinity protein purification [2]. Different point mutants are available, where selected amino acids that are supposed to be involved in DNA binding are replaced by alanine residues. The selection of these specific amino acid residues is based on the previously published X-ray crystal structure of the DNA:protein complex [3].

The consequences of DNA:protein complex formation are investigated by circular dichroism (CD) spectroscopy. Structural changes of the DNA upon binding of the PhoB (wild type) and several mutants can be observed. Using surface plasmon resonance measurements (SPR), the formation of DNA:protein complexes is investigated. Based on DNA molecules with two binding sites, the binding properties of the DBD PhoB towards DNA are analyzed [4]. Furthermore a cyclic PhoB mutant is generated by the use of the transpeptidase sortase A and will be analyzed in vitro and in vivo, too.

The data resulting from these studies provide insight into the molecular recognition of specific DNA sequences by proteins and their binding mechanisms.


References:


[1] A. Blanco, M. Sola, A. Gomis-Rüth, M. Coll, Structure2002, 10, 701-713.

[2] M. Xu, T. Evans, Methods2001, 24, 257-277.

[3] P. Bachhawat, G. Swapna, G. Montelione, A. Stock, Structure2005, 13, 1353-2363.

[4] K. Wollschläger, K. Gaus, N. Sewald, Small2009, 5, 484-495.


Acknowledgement:


This project is supported by the DFG (SFB 613).


List of Publications:


M. Ritzefeld, K.Wollschläger, G. Niemann, D. Anselmetti, N. Sewald, Minor groove recognition is important for the transcription factor PhoB: a surface plasmon resonance study, Mol. BioSyst., 2011, 7, 3132-3142.


Poster:


G. Niemann, M. Ritzefeld, K. Wollschläger, N. Sewald, “Interaction of DNA and DNA-binding proteins by the example of the transcription factor PhoB from E. coli”, 31th European Peptide Symposium, Copenhagen (05.-09-09.2010)

G. Niemann, M. Ritzefeld, K. Wollschläger, N. Sewald, “DNA-protein interactions between the transcription factor PhoB from E. coli and corresponding DNA sequences”, 10th German Peptide Symposium, Berlin (07.-10.03.2011)