Spectroscopic study of the MADS box
Barbora Řezáčová1,2*, Yves-Marie Coïc3, Christian Zentz2, Pierre-Yves Turpin2, Sergei Kruglik2, Václav Profant1 and Josef Štěpánek1
1 Institute of Physics, Charles University in Prague, Faculty of Mathematics and Physics, Ke Karlovu 5, CZ-12116 Prague 2 (Czech Republic)
2Laboratoire Acides nucléiques et biophotonique, FRE 3207, Université Pierre et Marie Curie, place Jussieu 2, 75252 Paris Cedex 5 (France)
3Unité de Chimie des Biomolécules, URA 2128 CNRS, Institut Pasteur, Rue Du Dr. Roux 2, 75724 Paris Cedex 15 (France)
The MADS box family of transcription factors plays an essential role in the gene regulation of higher organisms. Its acronym is derived from initials of four of the originally identified members of the family: MCM1, AG, DEFA and SRF [1]. These transcription factors share a highly conserved DNA sequence of 56 amino acids, called the MADS box.
The serum response factor (SRF) plays a crucial role in the activation of genes that respond to mitogenes and also in the regulation of muscle specific genes [2]. The SRF transcriptional activity is determined by its binding as a symmetric dimer to the so called CArG box element, one of the most vastly characterized DNA binding sequences recognized by the MADS box transcription factors. Preceding studies on 20-mer oligonucleotides bearing the specific high affinity CArG box of the c-fos enhancer, disclosed an equilibrium between bend and linear conformers [3] which is partly maintained even after the protein/DNA complex formation [4]. Still little is known about the recognition process, how targeted DNAs are recognized by these transcription factors. Thus the conserved MADS box motif may serve as a basic model for study of common functional properties of DNA recognition within the whole protein family (over 200 members).
The structural and dynamic properties of the MADS box and its shorter segments were studied by advanced spectroscopy techniques: fluorescence, UV absorption and Raman scattering. Acquired data were treated by a recently developed method based on factor analysis, the singular value decomposition. Owing to this method we were able to detect even very weak spectral changes. Vibrational spectra of the most probable conformers of shorter MADS box segments were simulated in Gaussian09 program suite in order to obtain the detailed interpretation of experimental data. Effects caused by variations of environmental conditions were also investigated. Especialy, a high sensitivity to pH (charged amino acids of MADS box i.e. tyrosine, lysine, and glutamic acid) was observed.
This work is supported by the Czech Science Foundation (project 202/09/0193), Grant Agency of Charles University (project 402111). B.R. gratefully acknowledges the French Government support for her stay in Laboratoire Acides nucléiques et biophotonique.
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