Hahn, StevenKamenova, Ivanka Dragomirova2013-11-142015-12-142013-11-142013Kamenova_washington_0250E_12229.pdfhttp://hdl.handle.net/1773/24221Thesis (Ph.D.)--University of Washington, 2013The core promoter is defined as the minimal sequence of DNA surrounding the transcription start site that is required for transcription of mRNA genes by the eukaryotic RNA Polymerase II (Pol II). Multiple core promoter elements exist in higher eukaryotes, and these DNA elements make distinct contributions to the combinatorial control of gene expression. The TATA box is the most ancient and conserved core promoter element, yet it is present in no more than 20% of Pol II promoters. To address the need for in vitro models of TATA-less genes, I used the Saccharomyces cerevisiae Ribosomal Protein gene promoter RPS5, which lacks a TATA box, to develop an in vitro transcription system. Using this system to characterize the requirements of yeast TATA-less promoters, I found that the RPS5 promoter was dependent on the coactivactor complex TFIID in addition to requiring the TATA-binding protein TBP for transcription. The general transcription factors (IIA, TBP/TFIID, IIB, IIF, IIE, IIH) and Pol II were insufficient to drive transcription from the RPS5 promoter, suggesting that additional proteins are needed for transcription of TATA-less genes. I further examined the requirement for DNA binding by TBP both in vitro and in vivo, demonstrating that TATA-less promoters can be transcribed by TBP deficient in DNA binding. TBP binds to two distinct sites on the RPS5 promoter. Mutational analysis of these binding sites revealed that they are dispensable for in vitro transcription, while replacement with GC-rich DNA is detrimental. My work has established an in vitro system with a TFIID-dependent TATA-less promoter and implicated a more relaxed mechanism of TBP binding on this type of promoter.application/pdfen-USCopyright is held by the individual authors.RNA Polymerase; Rpb5; TATA-binding protein; TATA-less; TFIID; transcriptionMolecular biologyBiochemistrymolecular and cellular biologyThesis