Molecular Mechanism of Gene Transcription Activation

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 DESCRIPTION (provided by applicant): We seek to define the molecular mechanisms controlling eukaryotic mRNA gene transcription by RNA Polymerase II. Defects in mRNA biosynthesis may lead to human maladies such as cancer, diabetes and obesity. Thus, it is imperative to dissect the very first step in gene expression, mRNA gene transcription, so that we can understand both normal and pathological states. Our ongoing work utilizes the genetically-tractable baker's yeast, Saccharomyces cerevisiae, as a model to elucidate the multiple roles that the highly evolutionarily-conserved transcription factor complex TFIID plays in mRNA gene transcription. TFIID is composed of 15 subunits (the TATA box Binding Protein, TBP, plus 14 TBP-associated factors, Taf1¿Taf14). TFIID is unique among the six so-called General Transcription Factors (GTFs: TFIIA, B, D, E, F, H) in that not only is it required for promoter-directed Pre-Initiation Complex (PIC) formation (indeed TFIID is the GTF that recognizes and binds the TATA promoter element), but TFIID also acts as a transcriptional coactivator on certain metazoan mRNA-encoding genes. TFIID is resident on, and its function is required for, the transcription of over 90% of mRNA encoding genes from yeast to humans. Our recent work has shown that yeast TFIID serves as a transcriptional coactivator for the transcription factor Repressor activator protein 1 (Rap1) via collaboration with TFIIA. Rap1 is an essential transfactor that activates transcription of the 100+ genes that encode the complement of proteins composing the ribosome; ribo- some levels and translation are universally rate-limiting for cellular proliferation. Null mutations of Rap1, RNA polymerase II and the six GTFs are all lethal, results indicating that all of these proteins contribute key cellular functions. We propos experiments that employ a multifaceted approach combining biochemical, genetic, proteomic, and structural methods to elucidate how the interplay of TFIID and Rap1 with each other, with enhancer- promoter DNA, and with GTFs and RNA Polymerase II, leads to the precise and controlled activation of gene transcription. Ultimately we plan to study this process on a natural, in vivo-assembled, mRNA-encoding gene. We believe our work will provide novel and global insights to understand mRNA gene transcription regulatory mechanisms.