Development of Computational Methods to Characterize Transcriptional Precision and Chromatin Dynamics in Yeast

TBP nucleates the assembly of the transcription pre-initiation complex. Although TBP can bind promoters with high stability in vitro, recent results establish that virtually the entire TBP population is highly dynamic in yeast nuclei in vivo. The dynamic behavior of TBP is a consequence of the enzymatic activity of the essential Snf2/Swi2 ATPase Mot1, suggesting that ensuring a highly mobile TBP population is critical for transcriptional regulation on a global scale. We studied the effect of altering TBP binding dynamics on transcription using tiling arrays and a new computational analysis method to determine how perturbed TBP dynamics impact the precision of RNA synthesis in Saccharomyces cerevisiae. We found that in mot1-42 cells RNA length changes were observed for 713 genes with prematurely terminated transcripts representing the largest class of events. Genetic and genomic analyses support the conclusion that these effects on RNA length are mechanistically tied to dynamic TBP occupancies at certain types of promoters. To further capture the TBP binding dynamics we developed a novel method based on modified ChIP assay with sub-second temporal resolution. The time dependence of formaldehyde crosslinking was utilized to extract in vivo on- and off-rates for site-specific chromatin interactions varying over a ~100-fold dynamic range. Using this method, we show that a novel regulatory process shifts weakly bound TATA-binding protein to stable promoter interactions, thereby facilitating transcription complex formation.