Purification and characterization of chicken ovalbumin upstream promoter transcription factor from HeLa cells

Abstract

A transcription factor which binds to the chicken ovalbumin upstream promoter (COUP) sequence spanning between -70 and -90 is required for efficient transcription of the ovalbumin gene. This COUP transcription factor has been purified approximately 200,000-fold by a combination of conventional column and sequence-specific DNA affinity column chromatography. A few polypeptides were identified in the purified preparation on sodium dodecyl sulfate gel. Upon renaturation, all the major polypeptides in the molecular size range between 43 and 53 kDa bound specifically to the COUP sequence. Furthermore, at least one of the renatured polypeptides in the region of 43-45 kDa retained transcriptional activity. The binding of the COUP transcription factor to the ovalbumin promoter cannot be competed by DNA fragments which contain the CCAAT box promoter sequence. Since the COUP and CCAAT binding proteins can be separated on an S300 column, they are distinct molecules. Using band-shifting assays and 3' and 5' deletion mutants and oligonucleotide mutants, the sequence important for binding was mapped.