We have recently purified, to homogeneity, the yeast equivalent of human general transcription factor TFIID. Unlike the human which exhibits a native MW of -150,000, the yeast factor is monomeric and apparently globular, being composed of a single polypeptide of Mr=27,000. The yeast factor is a DNA binding protein which interacts specifically with the TATA- sequence of the Adenovirus 2 Major Late Promoter (Ad2 MLP). More importantly, yeast TFIID efficiently promotes transcription in vitro of the Ad2 MLP. Protein sequencing of year TFIID was successfully performed. Synthetic oligonucleotides, derived from amino acid sequence information, were used to clone the gene encoding yeast TFIID (TF2D). We propose to dissect the TF2D gene, both genetically and biochemically, in order to elucidate the mechanisms by which TFIID mediates template selection, factor-factor interactions and enhancer-directed stimulation in transcription; all the reactions in which TFIID is known to participate (at least in vitro). We will use the Saccaromyces TF2D gene to clone homologous sequences from other lower eucaryotes. We will use both nucleic acid hybridization and genetic complementation methods to identify and isolate these genes. These heterologous genes will be examined in detail in order to elucidate conserved structural and functional domains of these proteins. Understanding the details of TFIID function will greatly expand our comprehension of eucaryotic RNAP II transcription mechanisms and thus of eucaryotic gene regulation in general.
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