MOLECULAR ARCHITECTURE OF THE HSP70 PROMOTER AFTER DELETION OF THE TATA BOX OR THE UPSTREAM REGULATION REGION

GAGA factor, TFIID, and paused polymerase are present on the hsp70 pro moter in Drosophila melanogaster prior to transcriptional activation, In order to investigate the interplay between these components, mutant constructs were analyzed after they had been transformed into flies o n P elements, One construct lacked the TATA box and the other lacked t he upstream regulatory region where GAGA factor binds, Transcription o f each mutant during heat shock was at least 50-fold less than that of a normal promoter construct. Before and after heat shock, both mutant promoters were found to adopt a DNase I hypersensitive state that inc luded the region downstream from the transcription start site, High-re solution analysis of the DNase I cutting pattern identified proteins t hat could be contributing to the hypersensitivity. GAGA factor footpri nts were clearly evident in the upstream region of the TATA deletion c onstruct, and a partial footprint possibly caused by TFIID was evident on the TATA box of the upstream deletion construct, Permanganate trea tment of intact salivary glands was used to further characterize each promoter construct, Paused polymerase and TFIID were readily detected on the normal promoter construct, whereas both deletions exhibited red uced levels of each of these factors, Hence both the TATA box and the upstream region are required to efficiently recruit TFIID and a paused polymerase to the promoter prior to transcriptional activation, In co ntrast, GAGA factor appears to be capable of binding and establishing a DNase I hypersensitive region in the absence of TFIID and polymerase , Interestingly, purified GAGA factor was found to bind near the trans cription start site, and the strength of this interaction was increase d by the presence of the upstream region, GAGA factor alone might be c apable of establishing an open chromatin structure that encompasses th e upstream regulatory region as well as the core promoter region, thus facilitating the binding of TFIID.