GENOMIC FOOTPRINTING OF THE HSP70 AND HISTONE H3 PROMOTERS IN DROSOPHILA EMBRYOS REVEALS NOVEL PROTEIN-DNA INTERACTIONS

The transcriptional potential of the hsp70 heat shock gene promoter is established prior to induction by stress, It has been shown previousl y that the TBP subunit of TFIID is associated with the TATA element an d that RNA polymerase II is paused downstream from the transcription s tart site, In order to identify new interactions involved in establish ing this potentiated state, a detailed analysis of the molecular archi tecture of a single copy of the hsp70 promoter was performed, A suitab ly marked promoter was stably integrated using P-element-mediated tran sformation so as to overcome any ambiguity that might be associated wi th analyzing the five copies of the endogenous gene, Genomic footprint ing using DNase I revealed two previously unidentified interactions, F irst, the GAGA element located at -120 is protected by protein, Second ly, the pattern of DNase I cleavage in the vicinity of the transcripti on start is found to bear significant similarity to the pattern associ ated with binding of purified TFIID, Noting that purified GAGA factor and TFIID interact similarly with the hsp70 and H3 promoters, the arch itecture of the endogenous H3 promoter was analyzed to determine what interactions might be needed to establish a potentiated state containi ng a paused polymerase. Despite the detection of TFIID and GAGA on the H3 promoter, no paused polymerase is evident, In addition, no protein s appear to interact with the transcription start, These results sugge st that the GAGA factor and TFIID are not sufficient to establish a po tentiated state containing paused polymerase and that TFIID interactio ns downstream from the TATA element could be important for pausing.