DNA and Protein Footprinting Analysis of the Modulation of DNA Binding by the N-Terminal Domain of the <i>Saccharomyces cerevisia</i>e TATA Binding Protein

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 singlestranded 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.Snf2/Swi2 enzymes play critical roles in regulation of essentially all DNA metabolic processes by utilizing the energy of ATP hydrolysis to alter protein-DNA interactions (1-4). Mot1, 2 a Snf2/Swi2 protein, dissociates the TATA-binding protein (TBP) from promoters and thereby regulates TBP distribution in the cell (5-9). By exploiting the Mot1 experimental system, biochemical analyses of the TBP-DNA dissociation reaction have provided general insight into mechanisms by which Snf2/Swi2 ATPases catalyze rearrangements of stable protein-DNA complexes (4). The N terminus of Mot1 interacts with TBP through its flexible HEAT repeats and in this way recruits Mot1 to TBP-DNA (7, 10 -14). The C-terminal region of Mot1, which comprises the ATPase domain, interacts with DNA upstream of the TATA box (13,14). Given the sequence and structural similarity among Snf2/Swi2 ATPase domains (15, 16), a central goal in the field has been to decipher the mechanisms by which these enzymes couple ATP hydrolysis to dissociation or rearrangement of protein-DNA complexes.The Snf2/Swi2 ATPases comprise a group within the SF2 helicase superfamily (17). Thus, their mechanisms are proposed to be similar to those of helicases except that they do not catalyze DNA strand separation (15,18). Nonetheless, the Snf2/ Swi2 family has well over a thousand members, and the great majority of them have not been characterized biochemically (19). Biochemical studies of helicases have revealed diverse mechanistic properties (3), suggesting that Snf2/Swi2 enz...

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism of Mot1, a TATA-binding Protein (TBP)-DNA Dissociating Enzyme

Viswanathan

True

Auble

2016

“…53 with the following modifications: 1 mM ␤-mercaptoethanol was included in all buffers. An additional wash step with 60 mM imidazole was included prior to elution, thrombin digestion was conducted at 12°C overnight, CaCl 2 was omitted from buffer F, and 5 mM imidazole was included in buffer F. TBP was concentrated (using an Amicon concentrator with 10,000 molecular weight cut-off) into the final storage buffer.…”

Section: Methodsmentioning

confidence: 99%

Two-step Mechanism for Modifier of Transcription 1 (Mot1) Enzyme-catalyzed Displacement of TATA-binding Protein (TBP) from DNA

Moyle-Heyrman

Viswanathan

Widom

et al. 2012

Background: Mot1 catalyzes ATP-dependent disruption of TBP-DNA complexes. Results: Mot1 binding to TBP-DNA complexes induces a conformational intermediate in which DNA is unbent. Conclusion: We propose a two-step mechanism for Mot1 in which DNA unbending is followed by ATP-dependent DNA translocation. Significance: Mot1 is a model Snf2/Swi2 enzyme whose catalytic mechanism is relevant for understanding how other enzymes in the family function.

“…Full-length yeast TBP and TBP core domain (TBPc) were isolated to apparent homogeneity using bacterial overexpression systems as described previously (39). A bacterial expression plasmid for production of TBP E188C was constructed using standard procedures (40).…”

Section: Methodsmentioning

confidence: 99%

“…A bacterial expression plasmid for production of TBP E188C was constructed using standard procedures (40). TBP E188C was expressed and purified as described for the wild-type protein (39).…”

Section: Methodsmentioning

confidence: 99%

Function and Structural Organization of Mot1 Bound to a Natural Target Promoter

Sprouse

Shcherbakova

Cheng

et al. 2008

Self Cite

Mot1 is an essential, conserved TATA-binding protein (TBP)-associated factor in Saccharomyces cerevisiae and a member of the Snf2/Swi2 ATPase family. Mot1 uses ATP hydrolysis to displace TBP from DNA, an activity that can be readily reconciled with its global role in gene repression. Less well understood is how Mot1 directly activates gene expression. It has been suggested that Mot1-mediated activation can occur by displacement of inactive TBP-containing complexes from promoters, thereby permitting assembly of functional transcription complexes. Mot1 may also activate transcription by other mechanisms that have not yet been defined. A gap in our understanding has been the absence of biochemical information related to the activity of Mot1 on natural target genes. Using URA1 as a model Mot1-activated promoter, we show striking differences in the way that both TBP and Mot1 interact with DNA compared with other model DNA substrates analyzed previously. These differences are due at least in part to the propensity of TBP alone to bind to the URA1 promoter in the wrong orientation to direct appropriate assembly of the URA1 preinitiation complex. The results suggest that Mot1-mediated activation of URA1 transcription involves at least two steps, one of which is the removal of TBP bound to the promoter in the opposite orientation required for URA1 transcription.The TATA-binding protein (TBP) 2 is a central component of the RNA polymerase II (pol II) preinitiation complex (PIC) (1-3). Transcription can be regulated during many different steps in PIC assembly, but the regulation of TBP binding to promoters is a fundamentally important step exploited for regulation of a large number of genes (4, 5). TBP is a saddle-shaped protein that interacts with DNA as a monomer (6, 7). Because TBP can straddle DNA in either of two directions, its binding polarity determines the orientation of the assembled PIC and hence the direction in which transcription initiates (8). TBP recruitment to promoters is regulated by chromatin, activators, and co-activators that contact TBP directly, and general factors that modulate TBP binding and activity (1, 2, 4, 9 -11).NC2 and Mot1 are essential, conserved regulators of TBP function that cooperate to regulate gene expression on a global scale (12)(13)(14). NC2, a heterodimer of the Bur6 and Ydr1 subunits in yeast (15), interacts with the TBP-DNA complex and can block subsequent steps in PIC assembly (16). NC2 is also a direct activator of gene expression (17) that is found at promoters in vivo in proportion to the level of transcription (14). How this apparent negative regulator of transcription functions as an activator is likely related to the recent observation that NC2 binding can allow TBP to relocalize along the DNA contour (18). Such mobilization of TBP may be critical for clearance of TBP from inactive but high affinity sites, and may facilitate selection of appropriate TATA sequences among a collection of binding sites along accessible promoter DNA. Mot1 is a member of the Snf2/Swi2 ATPa...