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. TFIID and GAGA factor interact with specific sequences. A molecule of RNA polymerase II has initiated transcription but phased in a location proximal to the promoter. Transcriptional induction by heat shock involves the transcriptional activator, HSF, that becomes associated with the promoter during heat shock. The long-term objective of this proposal is to understand the mechanisms that establish the transcriptional potential and transcriptional activation of the hps70 promoter. Mostlikely, these types of mechanisms are shared by many eucaryotic genes so the insights that emerge from this project will have significant impact on understanding expression of genes involved in cancer, viral infections, and development. This proposal focuses on understanding the basis for why polymerase pauses in the promoter-proximal region of many genes. There are five specific aims: 1) Identify pausing factors by fractionating a pausing-competent, cell-free system. 2) Determine if the CTD is required for pausing by using an RNA polymerase II-dependent transcription reaction. 3) Determine if TAFs are required for pausing by using a TFIID-TBP-dependent transcription reaction. 4) Investigate the effects of mutations in the core promoter region in intact cells. 5) Analyze protein-DNA interactions on the hsp70 promoter in vitro and in vivo through crosslinking. An important component of this proposal is a cell-free system that reconstitutes the paused polymerase in a manner that reflects the situation found in cells. The paused polymerase is detected by monitoring the pattern of permanganate reactivity on the DNA. Factors involved in pausing the polymerase will be isolated by fractionating the extract and reconstituting pausing. In vivo analyses of promoter proximal pausing will also be performed by analyzing mutant promoters that have been transformed back into flies. Finally, two protein-DNA crosslinking techniques will be used to determine how components of the transcription apparatus are integrated on the hsp70 promoter in vivo and in vitro.
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