Date of Award
Doctor of Philosophy (PhD)
The general transcription initiation factor IID plays a central role in transcriptional control as a direct target for a diverse array of gene-specific regulatory factors and as the only template-bound class II initiation factor. The work described in this thesis concerns itself with the molecular characterization of this transcription initiation factor, starting with the cloning of cDNAs from a variety of organisms, including human, encoding a protein that may substitute native TFIID fractions in DNA binding and in vitro basal transcription assays. Sequence comparisons identify important structural motifs in the protein. Further functional analyses lead to the realization that this protein is the TATA box binding subunit (TBP) of a multi-protein TFIID complex whose other constituents are required for activator-responsive in vitro transcription and are responsible for TFIID's characteristic DNA interactions around the initiation region. A variety of biochemical approaches are discussed leading to the identification of more than a dozen class II TBP-associated factors (TAFs) that as a whole make up the TFIID complex and its characteristic functions. A bacterial expression system allowing for the convenient purification of large amounts of recombinant protein in non-denaturing conditions is presented. This has allowed structural studies on TBP that have culminated in detailed X-ray crystallographic structures of TBP by itself or bound to the TATA box revealing unprecedented DNA distortions. The design of a convenient mutagenesis approach of TBP is presented that may lead to a more refined understanding of TBP and TAF functions. Cloning of a small subunit (TAF20) of the TFIID complex is taken as a starting point to chart protein-protein interactions within the complex. Furthermore, sequence homologies suggest possible functions for some TAFs and lead to a proposed revision of TFIID's functional role in eukaryotic chromatin and in mechanisms of transcriptional initiation and regulation.
Hoffmann, Alexander, "A Molecular Characterization of Human General Transcription Factor IID" (1994). Student Theses and Dissertations. 353.