C-->G base mutations in the CArG box of c-fos serum response element alter its bending flexibility. Consequences for core-SRF recognition.
By binding to the CArG box sequence, the serum response factor (SRF) activates several muscle-specific genes, as well as genes that respond to mitogens. The core domain of the SRF (core-SRF) binds as a dimer to the CArG box C-5C-4A-3T-2A-1T+1T+2A+3G+4G+5 of the c-fos serum response element (SREfos). However, previous studies using 20-mer DNAs have shown that the binding stoichiometry of core-SRF is significantly altered by mutations C-5-->G (SREGfos) and C-5C-4-->GG (SREGGfos) of the CArG box [A Huet, A Parlakian, M-C Arnaud, J-M Glandières, P Valat, S Fermandjian, D Paulin, B Alpert & C Zentz (2005) FEBS J272, 3105-3119]. To understand these effects, we carried out a comparative analysis of the three 20-mer DNAs SREfos, SREGfos and SREGGfos in aqueous solution. Their CD spectra were of the B-DNA type with small differences generated by variations in the mutual arrangement of the base pairs. Analysis by singular value decomposition of a set of Raman spectra recorded as a function of temperature, revealed a premelting transition associated with a conformational shift in the DNA double helices from a bent to a linear form. Time-resolved fluorescence anisotropy shows that the fluorescein reporter linked to the oligonucleotide 5'-ends experiences twisting motions of the double helices related to the interconversion between bent and linear conformers. The three SREs present various bent populations submitted, however, to particular internal dynamics, decisive for the mutual adjustment of binding partners and therefore specific complex formation.
Stepanek
Josef
J
Laboratoire de Biophysique Moléculaire Cellulaire & Tissulaire, Université Pierre et Marie Curie, Evry, France.
Vincent
Michel
M
Turpin
Pierre-Yves
PY
Paulin
Denise
D
Fermandjian
Serge
S
Alpert
Bernard
B
Zentz
Christian
C
D000818:Animals; D006801:Humans; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D027602:Serum Response Element / Q000235:genetics*; D026362:Serum Response Factor / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013059:Spectrum Analysis, Raman
D016760:Proto-Oncogene Proteins c-fos; D026362:Serum Response Factor