MOT1 can activate basal transcription in vitro by regulating the distribution of TATA binding protein between promoter and nonpromoter sites

MOT1 is an ATPase which can dissociate TATA binding protein (TBP)-DNA compl exes in a reaction requiring ATP hydrolysis. Consistent with this observati on, MOT1 can repress basal transcription in vitro. Paradoxically, however, some genes, such as HIS4, appear to require MOT1 as an activator of transcr iption in vivo. To further investigate the function of MOT1 in basal transc ription, we performed in vitro transcription reactions using yeast nuclear extracts depleted of MOT1, Quantitation of MOT1 revealed that it is an abun dant protein, with nuclear extracts from wild-type cells containing a molar excess of MOT1 over TBP, Surprisingly, MOT1 can weakly activate basal tran scription in vitro. This activation by MOT1 is detectable with amounts of M OT1 that are approximately stoichiometric to TBP, With amounts of MOT1 simi lar to those present in wild-type nuclear extracts, MOT1 behaves as a weak repressor of basal transcription. These results suggest that MOT1 might act ivate transcription via an indirect mechanism in which limiting TBP can be liberated from nonpromoter sites for use at promoters, In support of this i dea, excess nonpromoter DNA sequesters TBP and represses transcription, but this effect can be reversed by addition of MOT1, These results help to rec oncile previous in vitro and in vivo results and expand the repertoire of t ranscriptional control strategies to include factor-assisted redistribution of TBP between promoter and nonpromoter sites.