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Gene transcription is a central process of life and is highly regulated with a multitude of transcription factors. Recently, we could show that dynamics of the Tata-box Binding Protein (TBP) on the promoter site also plays a role in gene regulation. Using single-pair Forster Resonance Energy Transfer (spFRET) experiments, we have monitored the conformation of TBP bound to the H2B promoter site in the presence and absence of Mot1. Upon the binding of Mot1 to the TBP-DNA complex, a high FRET state between TBP and labeled DNA is formed. Contrary to what was expected, Mot1 bound to TBP-DNA complexes was unable to dissociate TBP from the DNA upon addition of ATP. Instead, the TBP-DNA complex returned to its original conformation even though Mot1 remain bound to the complex. Only when Mot1 was also present at nM concentrations in solution was dissociation of TBP from the promoter site observed upon addition of ATP. This suggests that a single Mot1 is either insufficient or inefficient at dissociating TBP alone.We also used spFRET to monitor the DNA conformation during the interaction of Mot1 with TBP-DNA. TBP-bound DNA was observed to fluctuate between three conformations. The FRET values of the conformation varied depending on the orientation of TBP on the promoter. Fluctuations between the same conformations were observed when Mot1 was bound to the TBP-DNA complex but with an altered kinetics. The results suggest that Mot1 primes incorrectly bound TBP for dissociation, preferably freeing promoter sites with an inappropriately bound TBP molecule.

SNF2/SWI2-related ATPases employ ATP hydrolysis to disrupt protein–DNA interactions, but how ATP hydrolysis is coupled to disruption is not understood. Here we examine the mechanism of action of MOT1, a yeast SNF2/SWI2-related ATPase that uses ATP hydrolysis to remove TATA binding protein (TBP) from DNA. MOT1 function requires a 17 bp DNA ‘handle’ upstream of the TATA box, which must be double stranded. Remarkably, MOT1-catalyzed disruption of TBP–DNA does not appear to require DNA strand separation, DNA bending or twisting of the DNA helix. Thus, TBP–DNA disruption is accomplished in a reaction apparently not driven by a change in DNA structure. MOT1 action is supported by DNA templates in which the handle is connected to the TATA box via single-stranded DNA, indicating that the upstream duplex DNA can be conformationally uncoupled from the TATA box. Combining these results with proposed similarities between SNF2/SWI2 ATPases and helicases, we suggest that MOT1 uses ATP hydrolysis to translocate along the handle and thereby disrupt interactions between TBP and 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.

A family of variants of the PRM promoter of lambda phage was constructed, bearing nine base pair substitutions in a stretch of the spacer DNA separating the contacted -10 and -35 regions. The substituted sequences were chosen for their potential to adopt structures different from those of average B-form DNA and thus to affect the interaction of RNA polymerase with the two contacted regions. Characterization of the promoters in vitro and in vivo provides additional support for the lack of specific contacts in the substituted spacer region and shows that a small change in the relative rotational orientation of the -10 and -35 regions is inconsequential to promoter function. However, a 2-3-fold reduction in promoter activity is observed with promoters bearing substitutions of nonalternating dG-dC base pairs in either orientation. This corroborates other studies indicating the anomalous behavior of such sequences and suggests that the structure of the spacer DNA can modulate promoter recognition.

Escherichia coli RNA polymerase contacts promoter DNA at two regions (the −10 and −35 regions) which are separated by a segment of spacer DNA. Previously we showed that base substitutions in the spacer DNA can affect promoter strength both in vitro and in vivo; these results were interpreted to reflect altered structural properties of the substituted DNAs. Here we provide experimental support for this interpretation. The pattern of cleavage of the promoters with Neurospora crassa endonuclease and the reactivity of their guanine residues with dimethyl sulfate (DMS) suggest that the structures of the spacer DNAs in the promoters with altered transcriptional activities are distinct. In addition, the binding of RNA polymerase to the latter promoters induces characteristic enhancements in the extent to which specific guanine residues in the spacer DNAs react with DMS. We propose that for these promoters the substitutions in the spacer DNAs have affected the relative orientation of the −10 and −35 regions. The observed differences in promoter activity then would reflect the requirement for realignment of these regions during the process of open complex formation; we postulate that two such realignments occur.

A series of promoters with nine base-pair substitutions in the spacer DNA separating the -10 and -35 regions was used to demonstrate that Escherichia coli RNA polymerase is sensitive to events affecting the spacer DNA--a region not directly contacted by the enzyme. The drugs distamycin A and netropsin specifically enhanced the rate of functional complex formation at a promoter bearing a substitution of nonalternating A-T base pairs. The effect is exerted at an early step in the RNA polymerase-promoter interaction. We hypothesize that a drug-induced structural alteration in the spacer DNA occurs, similar to that normally resulting from RNA polymerase binding. These findings are relevant to an understanding of potential mechanisms of transcription activation.

The available evidence suggests that during the process of formation of a functional or "open" complex at a promoter, Escherichia coli RNA polymerase transiently realigns the two contacted regions of the promoter, thus stressing the intervening spacer DNA. We tested the possibility that this process plays an active role in the formation of an open complex. Two series of promoters were examined: one with spacer DNAs of 15 to 19 base-pairs and a derivative for which the promoters additionally contained a one-base gap in the spacer, so as to relieve any stress imposed on the DNA. Consistent with an active role for the stressed DNA in driving open complex formation, we have found that for promoters with a 17-base-pair spacer, the presence of a gap leads to a delay in the formation of an open complex, at a step subsequent to the initial binding of RNA polymerase to the promoter. The results with the other gapped promoters rule out direct binding of RNA polymerase to the region of the gap and indicate an increased flexibility in the gapped DNA. As not all observations with the spacer length series of gapped and ungapped promoters can be interpreted in terms of an active role of the spacer DNA without additional assumptions, such a role must still be considered tentative.