Snf2/Swi2-related ATPase Mot1 drives displacement of TATA-binding protein by gripping DNA

Abstract

Mot1 is a conserved Snf2/Swi2-related transcriptional regulator that uses ATP hydrolysis to displace TATAbinding protein (TBP) from DNA. Several models of the enzymatic mechanism have been proposed, including Mot1-catalyzed distortion of TBP structure, competition between Mot1 and DNA for the TBP DNA-binding surface, and ATP-driven translocation of Mot1 along DNA. Here, DNase I footprinting studies provide strong support for a 'DNA-based' mechanism of Mot1, which we propose involves ATP-driven DNA translocation. Mot1 forms an asymmetric complex with the TBP core domain (TBPc)-DNA complex, contacting DNA both upstream and within the major groove of the TATA Box. Contact with upstream DNA is required for Mot1-mediated displacement of TBPc from DNA. Using the SsoRad54-DNA complex as a model, DNA-binding residues in Mot1 were identified that are critical for Mot1-TBPc-DNA complex formation and catalytic activity, thus placing Mot1 mechanistically within the helicase superfamily. We also report a novel ATP-independent TBPc displacement activity for Mot1 and describe conformational heterogeneity in the Mot1 ATPase, which is likely a general feature of other enzymes in this class.

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

“…2, C and E). Complete TBP dissociation was not expected in this assay because competitor DNA was absent, thus allowing TBP rebinding (36,43). Remarkably, experiments carried out in parallel in the absence of ATP or in the presence of ATP␥S showed a similar [Mot1]-dependent decrease in FRET (Fig.…”

“…Nevertheless, a sequence alignment with other enzyme family members reveals that many residues implicated in DNA binding are conserved in Mot1 (40), and mutational analysis indicates that at least some are functionally important for the catalytic action of Mot1 (43). Furthermore, biochemical results and structural data (35,43) place the Mot1 ATPase domain in close proximity to the upstream DNA segment required for TBP-DNA dissociation.…”

“…TBP, nevertheless, must remain closely associated with the DNA in this state, because DNA unbending in the absence of nucleotide is not accompanied by a decrease in TBP-DNA FRET (Figs. 2B and 3B) or by loss of protection against digestion by DNase I (43). In the presence of ATP␥S, Mot1 may undergo a conformational change in its nucleotide-binding domain, conferring somewhat decreased stability of the complex.…”

“…Mot1 also Unbends Short TBP-DNA Complexes-A simple explanation for the ability of Mot1 to unbend DNA is that the DNA conformation is altered by the interaction of the ATPase domain with the ϳ17-bp DNA segment upstream of bound TBP (35,43). To test this, dual-labeled DNA FRET was measured with 14-and 21-bp TATA-containing DNAs, which support TBP binding but were predicted to be too short for Mot1-mediated TBP-DNA dissociation.…”

“…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.…”