The necessary intricacy of gene expression in higher metazoans requires a myriad of positive and negative regulators for temporal and cell-specific control of transcription. Despite this complexity, the core promoter TATA- element binding protein, TBP, is a ubiquitous, highly-conserved central component of the general transcription machinery of all eukaryotic promoters. Recently, a Drosophila protein with striking homology to dTBP, dTBP-related factor (dTRF), was identified and found, surprisingly, to be highly tissue-specific. This raise the intriguing possibility that some transcriptional regulators may utilize core promoter complexes other than those containing TBP and TFIID to direct cell-type specific expression of genes. This research plan will investigate the potential role dTRF plays in tissue-specific transcriptional control. Specific objectives are: (1) Identification of a functional dTRF complex and its associated factors. Immunopurification and Far Western blot assays are proposed to isolate potential dTRF-associated factors. (2) Identification of dTRF-regulated target genes. A random site-selection is proposed to identify high affinity of dTRF recognition elements later used to determine functionality and to identify potential Drosophila genes under transcriptional control by dTRF. (3) Functional relevance of dTRF:promoter context through in vivo and in vitro analysis. Cell culture models of dTRF-mediated gene control will be tested by assay of reporter constructs with putative dTRF targets. In addition, biochemical characterization of dTRF-dependent transcription through cell-free reconstitution is proposed. The recapitulation of regulated transcription of a bona-fide dtrf-target gene could provide novel insights into mechanisms employed by metazoans for tissue-specific gene expression.
