Molecular analysis of the SNF2/SWI2 protein family member MOT1, an ATP-driven enzyme that dissociates TATA-binding protein from DNA.
MOT1 is an essential Saccharomyces cerevisiae protein and a member of the SNF2/SWI2 family of ATPases. MOT1 functions by removing TATA-binding protein (TBP) from DNA, and as a consequence, MOT1 can regulate transcription both in vitro and in vivo. Here we describe the in vivo and in vitro activities of MOT1 deletion and substitution mutants. The results indicate that MOT1 is targeted to TBP both in vitro and in vivo via amino acids in its nonconserved N terminus. The conserved C-terminal ATPase of MOT1 appears to contribute to TBP-DNA complex recognition in the absence of ATP, but it appears to function primarily during the actual ATP-dependent dissociation reaction. Chimeric proteins in which homologous portions of SNF2/SWI2 have been substituted for the MOT1 ATPase can bind to TBP-DNA complexes but fail to dissociate these complexes in the presence of ATP, suggesting that the specificity of action of MOT1 is also conferred by the C-terminal ATPase. ATPase assays demonstrate that the MOT1 ATPase is activated by TBP. Thus, MOT1 undergoes at least two conformational changes: (i) an allosteric effect of TBP that mediates the activation of the MOT1 ATPase and (ii) an ATP-driven "power stroke" that causes TBP-DNA complex dissociation. These results provide a general framework for understanding how members of the SNF2/SWI2 protein family use ATP to modulate protein-DNA interactions to regulate many diverse processes in cells.
Auble
D T
DT
Department of Biochemistry, University of Virginia Health Science Center, Charlottesville 22908, USA. dta4n@virginia.edu
Wang
D
D
Post
K W
KW
Hahn
S
S
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000596:Amino Acids / Q000502:physiology; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018628:Gene Dosage; D009154:Mutation; D009687:Nuclear Proteins; D010957:Plasmids; D011993:Recombinant Fusion Proteins; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000596:Amino Acids; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035181:TATA-Box Binding Protein; D014157:Transcription Factors; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C073676:MOT1 protein, S cerevisiae; C499333:SNF2 protein, S cerevisiae; D004265:DNA Helicases