Identification of amino acids essential for DNA binding and dimerization in p67SRF: implications for a novel DNA-binding motif.
The serum response factor (p67SRF) binds to a palindromic sequence in the c-fos serum response element (SRE). A second protein, p62TCF binds in conjunction with p67SRF to form a ternary complex, and it is through this complex that growth factor-induced transcriptional activation of c-fos is thought to take place. A 90-amino-acid peptide, coreSRF, is capable for dimerizing, binding DNA, and recruiting p62TCF. By using extensive site-directed mutagenesis we have investigated the role of individual coreSRF amino acids in DNA binding. Mutant phenotypes were defined by gel retardation and cross-linking analyses. Our results have identified residues essential for either DNA binding or dimerization. Three essential basic amino acids whose conservative mutation severely reduced DNA binding were identified. Evidence which is consistent with these residues being on the face of a DNA binding alpha-helix is presented. A phenylalanine residue and a hexameric hydrophobic box are identified as essential for dimerization. The amino acid phasing is consistent with the dimerization interface being presented as a continuous region on a beta-strand. A putative second alpha-helix acts as a linker between these two regions. This study indicates that p67SRF is a member of a protein family which, in common with many DNA binding proteins, utilize an alpha-helix for DNA binding. However, this alpha-helix is contained within a novel domain structure.
Sharrocks
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AD
Spemann Laboratories, Max-Planck-Institut für Immunobiologie, Freiburg, Germany.
Gille
H
H
Shaw
P E
PE
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000737:chemistry*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D026362:Serum Response Factor
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D026362:Serum Response Factor