High-Throughput Mutagenesis of a Yeast Promoter
The TATA binding protein (TBP) is essential for yeast transcription, but only ~15% of yeast promoters contain a canonical TATA sequence. Transcription of these promoters is generally dependent on a complex known as SAGA, while TATA-less promoters are generally dependent on the TFIID complex. Mutations to the TATA sequence in a SAGA-dependent promoter have detrimental effects on transcription, but less well-characterized is how mutations affect transcription from a TATA-less promoter. For example, will the addition of a TATA sequence to an otherwise TATA-less promoter increase transcription or make the promoter dependent on SAGA, or are these promoters dependent on TFIID even in the context of a TATA box? In this project, the student will create a large library of variants (on the order of 100,000) of a TATA-less yeast promoter and perform selections for their activity in chemostats. High-throughput sequencing will be used to calculate the rate of transcription of each variant. These data should help to elucidate the transcriptional role of the TATA sequence and inform how general transcription factors like TBP and TFIID interact with TATA-less promoters. The student will become familiar with genetic and molecular biology techniques and high-throughput DNA sequencing.