The goal of our research is to understand the molecular mechanisms controlling eukaryotic mRNA gene transcription by RNA Polymerase II (Pol II). A comprehensive understanding of transcriptional regulatory mechanisms is absolutely essential if we are to truly appreciate both normal and pathological processes and conditions and ultimately intervene to cure disease. Our work utilizes the genetically tractable Saccharomyces cerevisiae, or Baker's Yeast, as a model for elucidating the detailed role that the general transcription factor (GTF) TFIID plays in the complicated process of mRNA gene transcription by Pol II. Our planned experiments represent the outgrowth of our long-term study of TBP, the TATA-box Binding Protein, a critical protein factor that binds to the promoter of mRNA-encoding genes in order to set into motion the chain of events culminating in Pol II transcription and thus gene expression. A significant fraction of the total cellular TBP is only functional when associated with RNA Polymerase-specific collections of additional TBP binding proteins known as TBP Associated Factors (TAFs). We have shown that in yeast cells there are fourteen integral Pol II TAFs, or TAFIIs, that associate with TBP to form the TFIID multi-subunit complex. We and others, working both with yeast and mammalian cells, have shown that the TFIID complex is intimately involved in the regulatory events that control the transcription of certain mRNA encoding genes. Therefore, understanding the composition and function of TBP-TAF complexes will be key to elucidating how gene transcription is controlled at the molecular level. The approaches that we plan to use to analyze TFIID will be multi-faceted and will combine biochemical, biophysical and genetic techniques to examine the interplay of these factors with TBP, each other, additional GTFs, trans-activator proteins, promoter DNA, and RNA polymerase II. Successful completion of these experiments will greatly increase our understanding of the all-important process of mRNA gene transcription and hence disease. ? ?
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