Cooperative binding of TEF-1 to repeated GGAATG-related consensus elements with restricted spatial separation and orientation.
The human transcriptional enhancer factor (TEF) family includes TEF-1, TEF-3, TEF-4, and TEF-5. The TEFs share a highly conserved 68-amino acid TEA/ATTS DNA-binding domain, which binds to SV40 GT-IIC (GGAATG), SphI (AGTATG), SphII (AGCATG), and muscle-specific M-CAT (GGTATG) enhansons. We determined the optimal DNA-binding consensus sequence for TEF-1. Using a purified GST-TEF-1 fusion protein and a random pool of synthetic oligonucleotides, 31 independent clones were obtained after six rounds of binding site selection. DNA sequences analysis revealed that 16 clones contained direct repeats with a 3-bp spacer (DR3), and 15 clones contained a single binding site. The predominate consensus half-site was GGAATG (67%), and the other elements were of the form G(A)GA(T/C)ATG. The TEF-1 bound to the DR3 as a dimer in a cooperative manner. Cooperative binding was dependent on the spacing and orientation of the half-sites and was inhibited by deoxycholate treatment, providing evidence that protein-protein interactions were involved. The data suggest that TEF dimerization is important for its ability to modulate gene transcription.
Jiang
S W
SW
Endocrine Research Unit, Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
Desai
D
D
Khan
S
S
Eberhardt
N L
NL
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors