A consensus DNA-binding site for the androgen receptor.
We have used a DNA-binding site selection assay to determine a consensus binding sequence for the androgen receptor (AR). A purified fusion protein containing the AR DNA-binding domain was incubated with a pool of random sequence oligonucleotides, and complexes were isolated by gel mobility shift assays. Individually selected sites were characterised by nucleotide sequencing and compiled to give a consensus AR-binding element. This sequence is comprised of two 6-basepair (bp) asymmetrical elements separated by a 3-bp spacer, 5'-GGA/TACANNNTGTTCT-3', similar to that described for the glucocorticoid response element. Inspection of the consensus revealed a slight preference for G or A nucleotides at the +1 position in the spacer and for A and T nucleotides in the 3'-flanking region. Therefore, a series of oligonucleotides was designed in which the spacer and flanking nucleotides were changed to the least preferred sequence. Competition experiments with these oligonucleotides and the AR fusion protein indicated that an oligonucleotide with both the spacer and flanking sequences changed had greater than 3-fold less affinity than the consensus sequence. The functional activity of these oligonucleotides was also assessed by placing them up-stream of a reporter gene in a transient transfection assay and correlated with the affinity with which the AR fusion protein bound to DNA. Therefore, sequences surrounding the two 6-bp half-sites influence both the binding affinity for the receptor and the functional activity of the response element.
Roche
P J
PJ
Molecular Endocrinology Laboratory, Imperial Cancer Research Fund, London, United Kingdom.
Hoare
S A
SA
Parker
M G
MG
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001943:Breast Neoplasms; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D051381:Rats; D011944:Receptors, Androgen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D015345:Oligonucleotide Probes; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins; D004247:DNA