E, quantified proximity ratio of Mot1 titration in the absence of nucleotide (red circles), in the presence of 0.1–1 mM ATP (blue triangles), in the presence of 0.1–1 mM ATPS (purple squares) or in the absence of DNA, Mot1, and nucleotide (black dotted line). B–E, emission spectra (direct Cy5 excitation subtracted and normalized to total intensity) of pre-formed TBP-DNA complexes (by incubation of 5 nM TBPAtto532 with 60 nM 3-Cy5Gap DNA) titrated with Mot1 in the presence of no nucleotide (B), 0.1 mM ATP (C), or 1 mM ATPS (D).
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The acceptor fluorophore (Cy5) is covalently attached to the 3-end of the bottom strand complementary to the TATA box (3-Cy5Gap), which presumably allows the fluorophore free rotation in the gap. The donor fluorophore (Atto532) is attached via maleimide chemistry to a unique cysteine at position 61 of a mutant TBP (59).
The DNA template is the same as described in Fig. A, schematic illustration of TBP-DNA FRET system. TBP-DNA FRET system monitors TBP-DNA interaction. To guide the eye, data points were fit to a simple binding curve. Both concentrations yielded equivalent results. Shown are DNA without TBP and Mot1 (black dotted line), Mot1-TBP-DNA complex in the absence of nucleotide (red circles), in the presence of 0.1–1 mM ATP (blue triangles) or 0.1–1 mM ATPS (purple squares). E, quantification of proximity ratio for all conditions. B–E, emission spectra (normalized to total intensity) of 10 nM TGapC DNA and 25 nM TBP binary complexes titrated with Mot1 in the presence of no nucleotide (B), 0.1 mM ATP (C), or 1 mM ATPS (D). This DNA template is referred to as TGapC. The bottom strand complementary to the TATA box has a TAMRA (donor) fluorophore covalently attached to the 3-end and a Cy5 (acceptor) fluorophore at the 5-end. The DNA template is the same as described in the legend to Fig. A, schematic illustration of the duallabeled DNA FRET system. Based on these findings, a model is presented for Mot1 that links a DNA conformationalĬhange with ATP-induced DNA translocation.ĭual-labeled DNA FRET system detects Mot1-dependent unbending of TBP-DNA complexes. Mechanisms of chromatin remodeling enzymes. Interestingly, and in contrast to full-length Mot1, the isolated Mot1 ATPase domain binds DNA,Īnd its affinity for DNA is nucleotide-dependent, suggesting parallels between the Mot1 mechanism and DNA translocation-based Strikingly, in the absence of ATP, Mot1 acts to unbend DNA, whereas TBP remains closely associated with the DNA in a stable Native gel electrophoresis and FRET were used to determine how Mot1 affects the trajectory of DNA in the TBP-DNA complex. Several mechanisms for Mot1Īction have been proposed, but how it catalyzes TBP removal from DNA is unknown. Lifetime is limited by the ATP-dependent TBP displacement activity of the Snf2/Swi2 ATPase Mot1. The TBP-promoter DNA complex contains sharply bent DNA and its interaction
The TATA box binding protein (TBP) is a central component of the transcription preinitiation complex, and its occupancy atĪ promoter is correlated with transcription levels.
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