Fig. 3. Mot1p dissociates TBP from Mot1p–TBP–DNA complexes formed on 14mer TATA DNA via its ATPase function. (A) Left: 50 nM TBP was added to 50 nM 14Rox DNA (DNA alone, first 60 s blue trace, r = ∼0.11, sampled and labeled 1). Binding was monitored for 10 min (red trace) and sampled (labeled 2). Fifty nanomolar Mot1p was added to 50 nM TBP–DNA binary complex and anisotropy monitored, red t = 0 trace changing to green trace upon Mot1p addition, this reaction was sampled (labeled 6). Note that when the cuvette was accessed for addition of components a large spike in signal was generated. Blue trace (labeled 4) Mot1p added at ∼60 s measures anisotropy after the addition of Mot1p to 14Rox DNA alone. Right: 1 mM ATP was added to each reaction (see arrow labeled ATP) and anisotropy monitored with time; these reactions also sampled for EMSA (labeled 3, 5 and 7). (B) Shown is the fluorescence scan of EMSA fractionation of 10 µl aliquots of samples 1–7 from (A). (C and D) Reactions formed and analyzed exactly as in (A) and (B), except that 50 nM Mot1p K1301A protein was utilized.