Ch. Wu et al., Analysis of core promoter sequences located downstream from the TATA element in the hsp70 promoter from Drosophila melanogaster, MOL CELL B, 21(5), 2001, pp. 1593-1602
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.