Direct interaction of the t1 transactivation domain of the human glucocorticoid receptor with the basal transcription machinery

We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the r1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified'rl specifically inhibited transcription from a basal promoter derived from the CYCI gene and from the adenovirus 2 major late core promoter in a concentration-dependent manner. This inhibition or squelching was correlated with the transactivation activity of r1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of 'r1. In addition, no specific interaction between 'r1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the 'F1 transactivation domain of the human glucocorticoid receptor interacts directly with the general transcriptional apparatus through some target protein(s) that is distinct from the TATA-binding factor. Furthermore, this assay can be used to identify interacting factors, since after phosphoceliulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of r1 was found. The effects of glucocorticoid steroid hormones are medi-ated by an intracellular soluble receptor protein. The glu-cocorticoid receptor is a member of a large family o