MOT1 Can Activate Basal Transcription In Vitro by Regulating the Distribution of TATA Binding Protein between Promoter and Nonpromoter Sites

Abstract

MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA complexes in a reaction requiring ATP hydrolysis. Consistent with this observation, MOT1 can repress basal transcription in vitro. Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcription in vivo. To further investigate the function of MOT1 in basal transcription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1. Quantitation of MOT1 revealed that it is an abundant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP. Surprisingly, MOT1 can weakly activate basal transcription in vitro. This activation by MOT1 is detectable with amounts of MOT1 that are approximately stoichiometric to TBP. With amounts of MOT1 similar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription. These results suggest that MOT1 might activate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters. In support of this idea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1. These results help to reconcile previous in vitro and in vivo results and expand the repertoire of transcriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites.

ACKNOWLEDGMENTS

We thank Fred Winston, John Chicca, Frank Pugh, Joe Reese, members of the Auble and Weil laboratories, and the University of Virginia Sixes and Sevens Research Discussion Group for insightful discussions.

This work was supported by grants from the National Institutes of Health (GM55763 to D.T.A. and GM52461 to P.A.W.).