Nucleic Acids Research, 1989, Vol. 17, No. 3 1177-1195
© 1989
MOLECULAR BIOLOGY |
Point mutational analysis of the human c-fos serum response factor binding site
Division of Biological Research, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto 500 Sherbourne Street, Toronto, Ontario M4X 1K9, Canada
*To whom correspondence should be addressed
Received August 10, 1988. Revised January 3, 1989. Accepted January 3, 1989.
A series of point mutants were generated in the human c-fos dyad symmetry element (DSE), found within the c-fos serum response element, to study the sequence requirements for its interaction with the human HeLa cell serum response factor (SRF). Plasmids that contain base substitutions within a core CC(A/T)6GG motif in the center of the DSE did not-compete, or competed very poorly, with the wild-type c-fos DSE for formation of a specific SRF-DSE complex in vitro. The CC(A/T)6GG motif is not sufficient for maximal binding of SRF, as several plasmids that contain base substitutions in the sequences flanking this core motif competed either poorer or better than the wild-type c-fos DSE for SRF binding. Evidence is presented that supports the idea that SRF binds in a symmetrical fashion. Results of in vivo transient expression assays in HeLa cells suggest that negative regulation of c-fos transcription observed in serum-deprived cells is mediated through SRF binding to the DSE.
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