Point mutational analysis of the human c-fos serum response factor binding site.
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.
Leung
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S
Division of Biological Research, Ontario Cancer Institute, Toronto, Canada.
Miyamoto
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NG
D001483:Base Sequence; D001667:Binding, Competitive; D001790:Blood Physiological Phenomena; D004252:DNA Mutational Analysis / Q000379:methods; D004268:DNA-Binding Proteins / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000097:blood / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos; D012045:Regulatory Sequences, Nucleic Acid; D026362:Serum Response Factor
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D026362:Serum Response Factor