The studies of COUP transcription factor from HeLa cells: purification and partial characterization

A transcription factor COUP, which is responsible for the recognition of the Chicken Ovalbumin Upstream Promoter sequence between -70 and -90, is required for efficient transcription of the ovalbumin gene. This COUP combination of conventional column and sequence specific DNA affinity column chromatography. A few polypeptides were identified in the purified preparation on an SDS-gel. Upon renaturation, all the major polypeptides in the molecular size range between 43 to 53 kD bound specifically to the COUP sequence. Furthermore, at least one of the renatured polypeptides in the region of 43-45 kD retained transcriptional activity. Using band-shifting assays and 3' and 5' deletion mutants and oligonucleotide mutants, the sequence important for binding was mapped. Methylation and ethylation interference assays revealed the contact sites for the purine base and the phosphate backbone. These studies indicate that the COUP transcription factor interacts with the base residue in the major groove and wraps around the DNA helix in one full turn.