Activation of the MMTV LTR promoter by steroid hormones proceeds through the assembly of a transcription initiation complex composed of factors NFI/CTF, octamer binding protein, and TFIID. We have found that loading of NFI occurs constitutively on transiently transfected DNA, indicating that recruitment of some factors (NF1 requires a chromatin modification mechanism, whereas other factors (Oct-1) probably interact directly with receptor). To study the mechanism by which this process occurs, it is necessary to attempt reconstitution of this system in vitro. Steroid receptors are in low abundance in tissues and therefore difficult to purify. We have cloned steroid receptor cDNAs (glucocorticoid and vitamin D) into the adenovirus genome to form recombinant viruses which can infect cultured cells and express very high levels of functional receptors. These receptors have been partially purified, and shown to be highly active in specific DNA binding. We have further examined the mechanism of DNA binding by individually synthesizing the putative """"""""zinc finger peptides"""""""" from the rat glucocorticoid receptor. Circular dichroism experiments demonstrated a significant alteration in secondary structure in the presence of zinc that is pH dependent and correlates directly with DNA binding. DNA binding experiments established that single fingers can successfully compete with the intact DNA binding domain in a novel gel shift competition assay. Mutations in conserved cysteines fail to compete. Mutations in the CII finger establish that the final cysteine is required for DNA binding. The results suggest that a single synthetic """"""""finger peptide"""""""" is able to bind to DNA in a sequence specific manner.
