Mechanism of binding of serum response factor to serum response element.
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.
Huet
Alexis
A
Biologie moleculaire de la differenciation, Université Paris 7, Paris, France.
Parlakian
Ara
A
Arnaud
Marie-Claire
MC
Glandières
Jean-Marie
JM
Valat
Pierre
P
Fermandjian
Serge
S
Paulin
Denise
D
Alpert
Bernard
B
Zentz
Christian
C
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D003893:Desmin / Q000235:genetics; D019281:Dimerization; D005454:Fluorescence Polarization; D016762:Genes, fos; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D027602:Serum Response Element / Q000502:physiology*; D026362:Serum Response Factor / Q000235:genetics / Q000378:metabolism*; D012996:Solutions
D003893:Desmin; D009841:Oligonucleotides; D011994:Recombinant Proteins; D026362:Serum Response Factor; D012996:Solutions; D004247:DNA