Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster

TFIID recognizes multiple sequence elements in the hsp70 promoter of Drosop hila. Here, we investigate the function of sequences downstream from the TA TA element. A mutation in the initiator was identified that caused an eight fold reduction in binding of TFIID and a fourfold reduction in transcriptio n in vitro. Another mutation in the +24 to +29 region was somewhat less inh ibitory, but a mutation in the +14 to +19 region had essentially no effect. The normal promoter and the mutants in the initiator and the +24 to +29 re gion were transformed into flies by P element-mediated transformation. The initiator mutation reduced expression an average of twofold in adult flies, whereas the mutation in the +24 to +29 region had essentially no effect. I n contrast, a promoter combining the two mutations was expressed an average of sixfold less than the wild type. The results suggest that the initiator and the +24 to +29 region could serve overlapping functions in vivo. Prote in-DNA cross-linking was used to identify which subunits of TFIID contact t he +24 to +29 region and the initiator. No specific subunits mere found to cross-link to the +24 to +29 region. In contrast, the initiator cross-linke d exclusively to dTAF230. Remarkably, dTAF230 cross-links approximately 10 times more efficiently to the nontranscribed strand than to the transcribed strand at the initiator.