The long term goal of my research is to determine the mechanism by which a eucaryotic gene activator stimulates transcription by RNA polymerase II (pol II). GAL4 derivatives are well suited for this task because they stimulate transcription greater than 100-fold in mammalian cell culture and cell free transcription systems. The current hypothesis is that a GAL4 derivative, bound to its 17-bp recognition site, stimulates transcription by contacting an unidentified component of the general transcription machinery, bound at the TATA box; the intervening DNA loops out to permit the interaction. The contact is thought to facilitate assembly of an active pol II transcription complex. Preliminary evidence suggests that some component of the HeLa TFIID fraction is necessary for activation and could, in principle, be the direct target of the activator. Multiple GAL4 derivatives stimulate transcription synergistically; the synergy is not due to cooperative binding of the activators to their DNA sites.
The specific aims of this proposal are to: 1). Determine how an activator affects assembly of a pol II transcription complex. Complete and partial pol II transcription complexes will be formed using the general factors from HeLa cells; the effect of a GAL4 derivative on assembly of the complexes will be measured using gel filtration, gel mobility shift and DNase I footprinting assays. 2). Purify the protein in the HeLa TFIID fraction that allows the transcriptional machinery to respond to a GAL4 derivative. TFIID and GAL4 affinity chromatography will be used to purify the factor. Lambda gt 11 libraries will be probed with biotinylated TFIID and GAL4 derivatives to isolate clones encoding the protein. 3). Determine whether an activator interacts with the general transcription machinery via DNA looping. Electron microscopy will be employed to visualize looping directly. The ability of an activator to stimulate transcription when its binding site and the TATA box are positioned on different, but catenated, DNA molecules will be tested. 4). Determine the mechanism of transcriptional synergy. The ability of an activating region to act synergistically when multimerized and attached to a single DNA binding domain will be measured.
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