Direct interaction of the tau 1 transactivation domain of the human glucocorticoid receptor with the basal transcriptional machinery.
We have used a yeast (Saccharomyces cerevisiae) cell free transcription system to study protein-protein interactions involving the tau 1 transactivation domain of the human glucocorticoid receptor that are important for transcriptional transactivation by the receptor. Purified tau 1 specifically inhibited transcription from a basal promoter derived from the CYC1 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 tau 1. Recombinant yeast TATA-binding protein (yTFIID), although active in vitro, did not specifically reverse the inhibitory effect of tau 1. In addition, no specific interaction between tau 1 and yTFIID could be shown in vitro by affinity chromatography. Taken together, these results indicate that the tau 1 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 phosphocellulose chromatography of a whole-cell yeast extract, a fraction that contained an activity which selectively counteracted the squelching effect of tau 1 was found.
McEwan
I J
IJ
Centre for Biotechnology, NOVUM, Huddinge University Hospital, Sweden.
Wright
A P
AP
Dahlman-Wright
K
K
Carlstedt-Duke
J
J
Gustafsson
J A
JA
D000260:Adenoviruses, Human / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002474:Cell-Free System; D004268:DNA-Binding Proteins / Q000502:physiology; D005786:Gene Expression Regulation; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011965:Receptors, Glucocorticoid / Q000502:physiology*; D012441:Saccharomyces cerevisiae; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D011965:Receptors, Glucocorticoid; D035181:TATA-Box Binding Protein; D014157:Transcription Factors