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

Moyle-Heyrman, Georgette; Viswanathan, Ramya; Widom, Jonathan; Auble, David T.

doi:10.1074/jbc.m111.333484

Cited by 13 publications

(20 citation statements)

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“…We suggest that the non-equivalent roles of DNA bases at the same position along the DNA length but on opposite strands argue for strand specificity in Mot1 action. Prior work provided evidence for Mot1-induced DNA unbending (14,40), which was suggested to "prime" the TBP-DNA complex for dissociation (Fig. 7).…”

Section: Discussionmentioning

confidence: 94%

“…Probe-specific Difference in ATPase Conformation-To determine whether DNA sequence can influence ATPase conformation, we tested a DNA probe called Moyle6Fe, which was used previously (40) and contains the same TATA sequence as probe 6Fe, but the upstream DNA sequence is different. We also compared the effect of ADP-AlF 4 with ADP-BeF 3 , a "ground state" ATP analog (41,42), using each of these two DNA probes.…”

Section: Resultsmentioning

confidence: 99%

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

Viswanathan

True

Auble

2016

Journal of Biological Chemistry

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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...

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Section: Discussionmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

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

Viswanathan

True

Auble

2016

Journal of Biological Chemistry

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“…The Swi2/Snf2 ATPase domain of Mot1 contacts DNA ϳ17 bp upstream of TBP and translocates toward TBP, loosening its association of TBP with DNA. Last, the latch of Mot1 occupies the DNAbinding groove of TBP, displacing the Mot1-TBP complex from DNA and preventing TBP from rebinding DNA (7)(8)(9)(10).…”

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confidence: 99%

Mot1 Redistributes TBP from TATA-Containing to TATA-Less Promoters

Zentner

Henikoff

2013

Molecular and Cellular Biology

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The Swi2/Snf2 family ATPase Mot1 displaces TATA-binding protein (TBP) from DNA in vitro, but the global relationship between Mot1 and TBP in vivo is unclear. In particular, how Mot1 activates transcription is poorly understood. To address these issues, we mapped the distribution of Mot1 and TBP on native chromatin at base pair resolution. Mot1 and TBP binding sites coincide throughout the genome, and depletion of TBP results in a global decrease in Mot1 binding. We find evidence that Mot1 approaches TBP from the upstream direction, consistent with its in vitro mode of action. Strikingly, inactivation of Mot1 leads to both increases and decreases in TBP-genome association. Sites of TBP gain tend to contain robust TATA boxes, while sites of TBP loss contain poly(dA-dT) tracts that may contribute to nucleosome exclusion. Sites of TBP gain are associated with increased gene expression, while decreased TBP binding is associated with reduced gene expression. We propose that the action of Mot1 is required to clear TBP from intrinsically preferred (TATA-containing) binding sites, ensuring sufficient soluble TBP to bind intrinsically disfavored (TATA-less) sites.T ATA-binding protein (TBP) is a general regulator of eukaryotic transcription required for initiation by all three eukaryotic RNA polymerases (1). TBP lies at the center of a complex transcriptional network (2) and as such is regulated by a diverse array of factors. One such TBP-regulating protein is modifier of transcription 1 (Mot1), a highly conserved (3) and essential (4, 5) member of the Swi2/Snf2 ATPase family. Mot1 uses the energy derived from ATP hydrolysis to remove TBP from DNA (6), and the mechanism of action of Mot1 is perhaps the best understood of all Swi2/Snf2-like ATPases. Mot1 recognizes TBP from upstream, associating with TBP from above via its acidic loops. The Swi2/Snf2 ATPase domain of Mot1 contacts DNA ϳ17 bp upstream of TBP and translocates toward TBP, loosening its association of TBP with DNA. Last, the latch of Mot1 occupies the DNAbinding groove of TBP, displacing the Mot1-TBP complex from DNA and preventing TBP from rebinding DNA (7-10).The finding that Mot1 displaces TBP from DNA (6) led to the hypothesis that it would act primarily as a transcriptional repressor. However, numerous transcriptional profiling studies have established that loss of Mot1 both increases and decreases gene expression (4,5,(11)(12)(13)(14)(15)(16)(17)(18). Furthermore, loss of Mot1 has also been found to reduce TBP association with some promoters (11, 12, 18-21). Several models have been put forward to reconcile these findings with the well-characterized TBP-displacing activity of Mot1: (i) a preinitiation complex (PIC) remodeling model, wherein Mot1 alters the PIC conformation, presumably via interaction with TBP followed by ATP hydrolysis and translocation, to enhance transcriptional permissiveness (13); (ii) a PIC formation model, wherein the TBP-Mot1 complex nucleates an alternative, transcriptionally competent PIC (22); (iii) a nucleosome remodeling mode...

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“…As previously shown by the crystal structure of TBP-DNA, TBP bends the promoter DNA approximately 90° (Kim et al 1993). When Mot1 binds to TBP-DNA the FRET study showed that Mot1 straightened the bent DNA somewhat, potentially priming the TBP for dissociation (Moyle-Heyrman et al 2012). Importantly, this unbending of the DNA is in the absence of ATP.…”

Section: The Role Of Mot1 In Transcriptional Regulationsupporting

confidence: 55%

“…Importantly, this unbending of the DNA is in the absence of ATP. A model was proposed that after the unbending of DNA that Mot1 then went through multiple rounds of ATP hydrolysis to remove TBP from DNA (Sprouse et al 2008b, Moyle-Heyrman et al 2012). The current model of Mot1-mediated removal of TBP from DNA is illustrated in a simple cartoon in Figure 1.5.…”

Section: The Role Of Mot1 In Transcriptional Regulationmentioning

confidence: 99%

Transcriptional Regulation by Mot1 and Spt16

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

Cited by 13 publications

References 68 publications

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

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

Mot1 Redistributes TBP from TATA-Containing to TATA-Less Promoters

Transcriptional Regulation by Mot1 and Spt16

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