MOT1-catalyzed TBP-DNA disruption: uncoupling DNA conformational change and role of upstream DNA

Darst, Russell P.

doi:10.1093/emboj/20.8.2028

Cited by 37 publications

(106 citation statements)

References 51 publications

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“…Regardless, the binding of ATP is not sufficient for Mot1 to efficiently displace TBP from DNA. Consistent with published results (15,16,42) and Fig. 1B, addition of ATP allowed Mot1 to disrupt the TBP-DNA complex.…”

Section: Resultssupporting

confidence: 92%

“…A template containing a 1-nt gap was employed to satisfy the requirement that there be at least 17 bp upstream of the TATA box for efficient Mot1-catalyzed TBP displacement while also allowing fluorophore placement (Fig. 1A) (42). We refer to this gap-containing duplex DNA template as "Gap DNA."…”

Section: Resultsmentioning

confidence: 99%

“…Mot1 and Mot1C Purification-Mot1 was purified as described previously (42,44). The C-terminal domain of Mot1, consisting of amino acids 1254 -1967 (Mot1C), was amplified from Saccharomyces cerevisiae, cloned into the pMCSG7 vector containing an N-terminal His tag (52) and transformed into BL21(DE3) pLysS cells (Novagen).…”

Section: Methodsmentioning

confidence: 99%

“…Mot1-TBP complexes have relaxed sequence specificity for DNA (36), but the cause of this altered behavior is unclear. In contrast to other Snf2/Swi2 family members (37)(38)(39)(40)(41), full-length Mot1 has no detectable DNA binding affinity (16,36,42,43), and the ATPase activity of Mot1 is stimulated by TBP in vitro (44,45). However, biochemical evidence indicates that the ATPase domain is in close proximity to DNA when Mot1 is assembled with TBP-DNA (35,43), suggesting direct Mot1-DNA interaction.…”

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confidence: 99%

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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

Journal of Biological Chemistry

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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.

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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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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

Journal of Biological Chemistry

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“…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). To determine the roles of DNA bases and backbone contacts in Mot1-mediated catalysis, we tested a series of DNA templates with 3-base gaps in either strand (Fig.…”

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 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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Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes

Parkhurst¹,

Parkhurst²,

Powell³

et al. 2002

Biopolymers

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Time-resolved Förster resonance energy transfer (trFRET) has been used to obtain interdye distance distributions. These distributions give the most probable distance as well as a parameter, sigma, that characterize the width of the distribution. This latter parameter contains information not only on the flexibility of the dyes tethered to macromolecules, but on the flexibility of the macromolecules. Both the most probable interdye distance as well as sigma provide insight into DNA static bending and DNA flexibility. Time-resolved fluorescence anisotropy and static anisotropy measurements can be combined to provide a measure of the cone angle within which the tethered dyes appear to wobble. When this motion is an order of magnitude faster than the average lifetime that characterizes transfer, an average value of the dipolar orientational parameter kappa2 can be calculated for various mutual dye orientations. The resulting kappa2 distribution is very much narrower than the limiting values of 0 and 4, allowing more precise distances and distance changes to be determined. Static and time-resolved fluorescence data can be combined to constrain the analyses of DNA-protein kinetics to provide thermodynamic parameters for binding and for conformational changes along a reaction coordinate. The parameter sigma can be used to model multiple DNA-protein complexes with varying DNA bend angles in a global fitting of trFRET data. Such a global fitting approach has shown how the range of bends in single base DNA variants, when bound by the TATA binding protein (TBP), can be understood in terms of two limiting forms. Time-resolved FRET, combined with steady-state FRET, can be used to show not only how osmolytes affect the binding of DNA to proteins, but also how DNA bending depends on osmolyte concentration in the DNA-protein complexes.

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MOT1-catalyzed TBP-DNA disruption: uncoupling DNA conformational change and role of upstream DNA

Cited by 37 publications

References 51 publications

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

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

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

Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes

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