Abstract
The essential Saccharomyces cerevisiae ATPase Mot1 globally regulates transcription by impacting the genomic distribution and activity of the TATA-binding protein (TBP). In vitro, Mot1 forms a ternary complex with TBP and DNA and can use ATP hydrolysis to dissociate the TBP-DNA complex. Prior work suggested an interaction between the ATPase domain and a functionally important segment of DNA flanking the TATA sequence. However, how ATP hydrolysis facilitates removal of TBP from DNA is not well understood, and several models have been proposed. To gain insight into the Mot1 mechanism, we dissected the role of the flanking DNA segment by biochemical analysis of complexes formed using DNAs with short single-stranded gaps. In parallel, we used a DNA tethered cleavage approach to map regions of Mot1 in proximity to the DNA under different conditions. Our results define non-equivalent roles for bases within a broad segment of flanking DNA required for Mot1 action. Moreover, we present biochemical evidence for two distinct conformations of the Mot1 ATPase, the detection of which can be modulated by ATP analogs as well as DNA sequence flanking the TATA sequence. We also show using purified complexes that Mot1 dissociation of a stable, high affinity TBP-DNA interaction is surprisingly inefficient, suggesting how other transcription factors that bind to TBP may compete with Mot1. Taken together, these results suggest that TBP-DNA affinity as well as other aspects of promoter sequence influence Mot1 function in vivo.
Highlights
With TATA-binding protein (TBP) through its flexible HEAT repeats and in this way recruits Mot1 to TBP-DNA (7, 10 –14)
The combined results suggest that ATPdriven TBP-DNA dissociation by Mot1 involves at least two distinguishable steps: the conversion of an open ATPase state to the closed state induced by ATP binding and short range DNA translocation, which occurs subsequent to ATP hydrolysis and probably coincides with ADP or Pi release
Affinity of Mot1 for Gapped DNA Complexes—To investigate how the Mot1 ATPase utilizes DNA in the TBP-DNA dissociation reaction, functional studies focused on the DNA segment upstream of the TATA sequence, which had been shown in prior work to be important for Mot1 action [32, 34]
Summary
Affinity of Mot for Gapped DNA Complexes—To investigate how the Mot ATPase utilizes DNA in the TBP-DNA dissociation reaction, functional studies focused on the DNA segment upstream of the TATA sequence, which had been shown in prior work to be important for Mot action [32, 34]. Top Strand DNA Is Required Subsequent to Reorganization of the ATPase Domain— bases close to the TATA box were important for the ATP-dependent displacement of TBP (e.g. gap probe 1), there was no difference in the Mot cleavage pattern with and without ADP-AlF4 using probe 10Fe in which FeBABE molecules were positioned within this same region of DNA. The new cleavage site induced by ADP-AlF4 was present in complexes formed on probe 6gapFe as well as probe 6Fe (Fig. 4C, lane 2 versus lane 1, arrow) This result suggests that the top strand DNA specified by the gap is required at a catalytic step subsequent to the nucleotide-induced conformational change in the Mot ATPase. The results further support the suggestion that DNA sequence variation of the Moyle6Fe probe somehow facilitates closed complex formation even in the absence of nucleotide
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