TATA-Binding Protein Variants that Bypass the Requirement for Mot1 In Vivo

Mot1 is an essential TATA-binding protein (TBP)-associated factor and Snf2/Swi2 ATPase that both represses and activates transcription. Biochemical and structural results support a model in which ATP binding and hydrolysis induce a conformational change in Mot1 that drives local translocation along DNA, thus removing TBP. While this activity explains transcriptional repression, it does not as easily explain Mot1-mediated transcriptional activation, and several different models have been proposed to explain how Mot1 activates transcription. To better understand the function of Mot1 in yeast cells in vivo, particularly with regard to gene activation, TBP mutants were identified that bypass the requirement for Mot1 in vivo. Although TBP has been extensively mutated and analyzed previously, this screen uncovered two novel TBP variants that are unique in their ability to bypass the requirement for Mot1. Surprisingly, in vitro analyses reveal that rather than having acquired an improved biochemical activity, one of the TBPs was defective for interaction with Pol II preinitiation complex (PIC) components and other regulators of TBP function. The other mutant was defective for DNA binding in vitro, yet was still recruited to chromatin in vivo. These results suggest that Mot1-mediated dissociation of TBP (or TBP-containing complexes) from chromatin can explain the Mot1 activation mechanism at some promoters. The results also suggest that PICs can be dynamically unstable, and that appropriate PIC instability is critical for the regulation of transcription in vivo. Poly (A)+ mRNA was harvested from WT MOT1, mot1-14, and the bypass strains TBP-Y185C and TBP-F207L to determine the effect of the mutant TBP alleles on transcription. The reference sample for each of the three mutants (mot1-14, TBP-Y185C, and TBP-F207L) was from WT MOT1 cells. The genes misregulated by TBP-Y185C and TBP-F207L were compared to those misregulated by mot1-14 to determine which Mot1-regulated genes were restored to WT expression levels in the bypass strains, and also to determine the subset of genes that were newly misregulated by each of the bypass mutants. Each comparison was conducted in duplicate, employing a fluor reversal. For each array, the data shown is the ratio of Mutant/WT for each probe.