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

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 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...

Transcriptional Regulation by Mot1 and Spt16

“…Genome-wide localization has also shown that Mot1 is predominately localized to promoters of genes it represses and activates, suggesting that Mot1 has a direct role in both types of regulation. This localization coincides with TBP localization at the promoters, and Mot1 has been shown to approach TBP from the upstream DNA, which is consistent with studies showing that the upstream DNA is required for Mot1 function and the results from Chapter II (Darst et al 2001, Wollmann et al 2011, Zentner and Henikoff 2013, Viswanathan et al 2016.…”

“…As already stated, the remaining sequence was exactly the same as the 5' Bio duplex. We then repeated the experiment to determine if TBP or (Maruta et al 1993, Viswanathan 2013, Viswanathan et al 2016). We determined if there was any change in the ATPase conformation between binding of ATP (as mimicked by the ground state ADP-BeF 3 ) and hydrolysis (as mimicked by the transition state ADP-AlF 4 ) (Fisher et al 1995, Liu et al 2008.…”

“…Here we showed the presence of two conformations of Mot1 based on our FeBABE approach (Viswanathan et al 2016). Earlier it was suggested that Mot1 could adopt two different conformations based on results that showed two different types of Mot1-TBP-DNA complexes (Sprouse et al 2008b).…”

“…This requirement of the upstream DNA was further supported by FeBABE experiments that showed that the Swi2/Snf2 ATPase domain contacted the upstream DNA (Wollmann et al 2011). An ATP analog was shown to capture a conformational intermediate in the Mot1 hydrolytic cycle (Viswanathan 2013, Viswanathan et al 2016). Previously, we had proposed that Mot1 binding to the upstream DNA could be affected by the DNA sequence (Sprouse et al 2008b).…”

Conformational changes and catalytic inefficiency associated with Mot1-mediated TBP–DNA dissociation

“…Indeed, Mot1 overexpression inhibits cell growth due to enhanced and unbalanced activity toward TBP (14,43). Consistent with this, recent evidence has suggested that Mot1 does not efficiently dissociate high affinity TBP–DNA complexes (44) and, in addition, Mot1 has been implicated in the turnover of nonspecific or transcriptionally inactive interactions with DNA (45).…”