Oestrogen receptor facilitates the formation of preinitiation complex assembly: Involvement of the general transcription factor TFIIB

The action of oestrogen hormones is mediated through the oestrogen receptor (ER), a member of a large superfamily of nuclear receptors that function a s ligand-activated transcription factors. Sequence-specific transcription f actors, including the nuclear receptor superfamily, are thought to interact either directly or indirectly with general transcription factors to regula te transcription. Although numerous studies have focused on the identificat ion of potential co-activators interacting with isolated trans-activation d omains of ER, few have investigated the mechanisms by which ER transmits it s signal to the basal transcription machinery. We show that ER does not sta bilize the binding of the TATA-box binding protein (TBP) of the TFIID compl ex, or of TFIIB to the promoter, although a stable ER-TBP-TFIIB-promoter co mplex was detected, suggesting that ER, TBP and TFIIB might interact with e ach other to form a complex to the promoter. We also demonstrate that ER bi nds specifically to TFIIB, a key component of the preinitiation complex. Af finity chromatography with immobilized deletion mutants of ER maps a TFIIB interaction region that encompasses the DNA-binding domain. The addition of excess TFIIB to transcription reactions in vitro did not, however, affect the magnitude of transcriptional activation by ER. These results indicate t hat, in contrast with current models, ER does not activate transcription by increasing the rate of assembly of TFIIB into the transcription complex. A n increased concentration of TFIIB was unable, by itself, to overcome the r equirement for ER. By using an immobilized promoter-template assay employin g nuclear extract from HeLa cells, recombinant human ER increased the stabl e association of subsequent components of the transcription machinery (TFII E and TFIIF), in correlation with ER-induced transcription. Our results sug gest that ER acts, in an early step, during or immediately after the format ion of template-committed complexes containing TFIIB, favouring the recruit ment of one or more components of the basic transcription machinery as well as co-activators.