Abstract
The minimal promoter of the Drosophila hsp70 gene contains a TATA box and two nonidentical HSE sequences, HSEI and HSEII, that synergistically activate the promoter. We have examined stereospecific alignment and spatial constraints in this promoter. Similar to deletion of HSEII, insertion in the spacer between the HSEs of 1 to 5 or 11 to 14 nucleotides (nt) reduced promoter activity to about 10%. In contrast, HSEII was capable of contributing to promoter activity when the spacer was either shortened by 2 or 4 nt or extended by 6 to 10 or 16 or 18 nt. Hence, half of the possible helical arrangements of HSEs are compatible, whereas the other half are essentially incompatible with efficient promoter function. HSEII was ineffective when its distance to HSEI was increased by more than 18 nt. In vitro, HSEII is a weak and HSEI a strong binding site for heat shock transcription factor HSF, and HSF binds to HSEII cooperatively. To find out whether the above periodicity reflects cooperative binding of HSF in vivo or represents the need of stereoalignment for synergistic activation of transcription, the weak HSF binding site HSEII was replaced with the strong binding site HSEI. This substitution greatly attenuated promoter periodicity, suggesting that the periodic effects are caused by cooperative binding of HSF to HSEII, and that stereoalignment of HSEs is not required for transcription activation. In agreement, in vitro assays using spacer mutants revealed cooperative binding of purified, recombinant HSF to HSEII with a similar periodicity as observed in vivo. Changing the distance between TATA and the HSEs did not produce promoter periodicity, indicating that stereoalignment of these elements is not important.
Highlights
The minimal promoter of the Drosophila hsp70 gene hsp genes from Drosophila and other organisms
To avoid confounding spacersequence effects, many of the substitutionpromoters received the same spacersequences that hadbeen used in theprevious sets of constructs(-lS, OS, 3s to 8S, and 14s to 18s; compare sequences in Fig. lB, Table I and Fig. 4B).Comparison of the activities of insertion promoters containingHSEII or the HSEI substitution revealed that theperiodic effects were greatly attenuated in the substitutiopnromoters: a 1-nt deletion reduced activity only by about 55% in the substitution promoter (Fig. 4A)but by 90%in the promoter containing HSEII (Fig. lA)
The maindifference between promoters with inverted and un-inverted HSEII was that the activities of +8 to +10 insertions were considerably lower in the former than in the latter promoters. These results suggest that the initicaolntact in the cooperative binding reactionmay not occur at theperfect motif but perhapsat the proximal end of HSEII
Summary
Plasmid Constructions-All constructs were derived frompD88 [12]. (3 x IO6 cells/plate) and were infected with recombinant virus at an. (3 x IO6 cells/plate) and were infected with recombinant virus at an In this construct, a Drosophila melanogaster hsp gene segment that multiplicity of infection of 10.Cells were heat-treated for 20 min at included, following an XhoI linker, 88 n t of 5'-nontranscribed sequence, 37 "C 48 h after infection, and nuclear extract was prepared as dethe entireRNA leader region, and thefirst seven hsp70codons is linked scribed [25] except that 0.25 mM ~-l-tosylamido-2-phenylethyclhloroin-frame t o a truncated P-galactosidase gene. Fragments were recovered from gel slices by heating a t plicate cultures transfected with construDc8t8 and with up t2o0 other 65 "C for 10 min, followed by phenol extraction and ethanol precipitaconstructs as well as mock-transfected cells.
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