Mot1 regulates the DNA binding activity of free TATA-binding protein in an ATP-dependent manner.
Mot1 is an essential Snf2/Swi2-related Saccharomyces cerevisiae protein that binds the TATA-binding protein (TBP) and removes TBP from DNA using ATP hydrolysis. Mot1 functions in vivo both as a repressor and as an activator of transcription. Mot1 catalysis of TBP.DNA disruption is consistent with its function as a repressor, but the Mot1 mechanism of activation is unknown. To better understand the physiologic role of Mot1 and its enzymatic mechanism, MOT1 mutants were generated and tested for activity in vitro and in vivo. The results demonstrate a close correlation between the TBP.DNA disruption activity of Mot1 and its essential in vivo function. Previous results demonstrated a large overlap in the gene sets controlled by Mot1 and NC2. Mot1 and NC2 can co-occupy TBP.DNA in vitro, and NC2 binding does not impair Mot1-catalyzed disruption of the complex. Residues on the DNA-binding surface of TBP are important for Mot1 binding and the Mot1.TBP binary complex binds very poorly to DNA and does not dissociate in the presence of ATP. However, the binary complex binds DNA well in the presence of the transition state analog ADP-AlF(4). A model for Mot1 action is proposed in which ATP hydrolysis causes the Mot1 N terminus to displace the TATA box, leading to ejection of Mot1 and TBP from DNA.
Darst
Russell P
RP
Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia 22908, USA.
Dasgupta
Arindam
A
Zhu
Chunming
C
Hsu
Jer-Yuan
JY
Vroom
Amy
A
Muldrow
Tamara
T
Auble
David T
DT
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035381:TATA-Binding Protein Associated Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D035181:TATA-Box Binding Protein / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C073676:MOT1 protein, S cerevisiae; D004265:DNA Helicases