The hsp70 heat shock gene in Drosophila is associated with a collection of nonhistone chromosomal proteins that establish and maintain the transcriptional potential of the gene before induction by heat shock or other stress. RNA polymerase II and TFIID are among this collection of proteins. the RNA polymerase II appears to have initiated transcription but arrested elongation approximately 25 nucleotides downstream of the transcription start. The long-term objective of this proposal is to understand the pathway of interactions that is responsible for this potentiated state. The types of interactions that are involved might play a key role in establishing the patterns of gene expression during early stages in development and for maintaining the transcriptional potential of a gene until induction occurs at an appropriate stage. The project has four specific aims: 1) Determine the molecular architecture of a normal hsp70 promoter that has been transformed into Drosophila. 2) Identify sequences that are essential for the potentiated state by determining the molecular architecture of mutant hsp70 promoters that have been transformed into flies. 3) Characterize protein-DNA and protein-protein interactions that occur during reconstitution of protein-DNA complexes on the hsp70 promoter. 4) In vitro transcription analysis of normal and mutant hsp70 promoters. P element-mediated transformation will be used to introduce single copies of normal and mutant hsp70 promoters into Drosophila. Genomic footprinting and protein-DNA crosslinking will be used to identify protein-DNA interactions and a nuclear run-on assay will be used to determine if RNA polymerase II is transcriptionally engaged on the promoters. Biochemical studies implicate 4 regions of the promoter that may be involved. These are the TATA element, the start site, a conserved sequence element centered at +25 and GAGA elements that are interdigitated with the heat shock regulatory elements. The molecular genetic analysis will be complemented with biochemical studies of highly purified protein fractions isolated from the nuclear extract of uninduced Drosophila embryos. In vitro and in vivo analysis with normal and mutant promoters should provide a comprehensive understanding of the potentiated state.
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