Purification and enzymic properties of Mot1 ATPase, a regulator of basal transcription in the yeast Saccharomyces cerevisiae

The 1867-residue Mot1 protein is a member of a superfamily of ATPases, some of which are helicases, that interact with protein-nucleic acid assemblies . Mot1 is an essential regulator of RNA polymerase II-dependent transcripti on in vivo and dissociates TATA box-binding protein (TBP)-DNA complexes in vitro. Mot1-(His)(6) was purified to apparent homogeneity from yeast extrac ts. The preparation efficiently dissociated TBP TATA complexes, suggesting that no other protein or cofactor is required. Mot1 behaved as a non-globul ar monomer in hydrodynamic studies, and no association was detected between differentially tagged co-expressed Mot1 constructs. ATPase activity was st imulated about 10-fold by high ionic strength or alkaline pH, or by deletio n of the N-terminal TBP-binding segment, suggesting that the N-terminal dom ain negatively regulates the C-terminal ATPase domain (Mot1C). Correspondin gly, at moderate salt concentration, Mot1 ATPase (but not Mot1C) was stimul ated greater than or equal to 10-fold by yeast TBP, suggesting that interac tion with TBP relieves a conformational constraint in Mot1. Double- or sing le-stranded TATA-containing DNA did not affect ATPase activity of Mot1 or M ot1C, with or without TBP. Mot1 did not exhibit detectable helicase activit y in strand displacement assays using substrates with flush ends or 5'- or 3'-overhangs. Mot1-catalyzed dissociation of TBP from DNA was not prevented by a psoralen cross-link positioned immediately preceding the TATA sequenc e. Thus, Mot1 most likely promotes release of TBP from TATA-containing DNA by causing a structural change in TBP itself, rather than by strand unwindi ng.