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Mechanism of binding of serum response factor to serum response element
Author(s) -
Huet Alexis,
Parlakian Ara,
Arnaud MarieClaire,
Glandières JeanMarie,
Valat Pierre,
Fermandjian Serge,
Paulin Denise,
Alpert Bernard,
Zentz Christian
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04724.x
Subject(s) - serum response factor , serum response element , oligonucleotide , rotational correlation time , chemistry , biophysics , dimer , tetramer , microbiology and biotechnology , fluorescence anisotropy , transcription factor , dna , biology , biochemistry , gene , molecule , organic chemistry , membrane , enzyme
Serum response factor (SRF) is a MADS transcription factor that binds to the CArG box sequence of the serum response element (SRE). Through its binding to CArG sequences, SRF activates several muscle‐specific genes as well as genes that respond to mitogens. The thermodynamic parameters of the interaction of core‐SRF (the 124–245 fragment of serum response factor) with specific oligonucleotides from c‐fos and desmin promoters, were determined by spectroscopy. The rotational correlation time of core‐SRF labeled with bis‐ANS showed that the protein is monomeric at low concentration (10 −7 m ). The titration curves for the fluorescence anisotropy of fluorescein‐labeled oligonucleotide revealed that under equilibrium conditions, the core‐SRF monomers were bound sequentially to SRE at very low concentration (10 −9 m ). Curve‐fitting data showed also major differences between the wild‐type sequence and the oligonucleotide sequences mutated within the CArG box. The fluorescence of the core‐SRF tyrosines was quenched by the SRE oligonucleotide. This quenching indicated that under stoichiometric conditions, core‐SRF was bound as a dimer to the wild‐type oligonucleotide, and as a monomer or a tetramer to the mutant oligonucleotides. Far‐UV CD spectra indicated that the flexibility of core‐SRF changed profoundly upon its binding to its specific target SRE. Lastly, the rotational correlation time of fluorescein‐labeled SRE revealed that formation of the specific complex was accompanied by a change in the SRE internal dynamics. These results indicated that the flexibility of the two partners is crucial for the DNA–protein interaction.