Mot1 is an essential yeast Snf2/Swi2-related ATPase that exerts both positive and negative effects on gene expression. In vitro, Mot1 can disrupt TATA-binding protein–DNA complexes in an ATP-dependent reaction. This activity can explain Mot1-mediated transcriptional repression, but how Mot1 activates transcription is unknown. We demonstrate that, remarkably, Mot1 is localized in vivo to promoters for both Mot1-repressed and Mot1-activated genes. Moreover, Mot1 ATPase activity is required for both activation and repression of gene activity. These findings suggest a novel function for the Mot1 ATPase at activated genes, perhaps involving ATP-driven reorganization of the preinitiation complex. Mot1 regulates the expression of ≈3% of yeast genes in cells grown in rich medium. Most of these genes are repressed by Mot1, consistent with Mot1's ATP-dependent TATA-binding protein–DNA dissociating activity. Additionally, ≈77% of the Mot1-repressed genes are involved in the diauxic shift, stress response, mating, or sporulation. The gene sets controlled by NC2 and Srb10 are strongly correlated with the Mot1-controlled set, suggesting that these factors cooperate in transcriptional control on a global scale

SNF2/SWI2-related ATPases employ ATP hydrolysis to disrupt protein±DNA interactions, but how ATP hydrolysis is coupled to disruption is not understood. Here we examine the mechanism of action of MOT1, a yeast SNF2/SWI2-related ATPase that uses ATP hydrolysis to remove TATA binding protein (TBP) from DNA. MOT1 function requires a 17 bp DNÀ handle' upstream of the TATA box, which must be double stranded. Remarkably, MOT1-catalyzed disruption of TBP±DNA does not appear to require DNA strand separation, DNA bending or twisting of the DNA helix. Thus, TBP±DNA disruption is accomplished in a reaction apparently not driven by a change in DNA structure. MOT1 action is supported by DNA templates in which the handle is connected to the TATA box via single-stranded DNA, indicating that the upstream duplex DNA can be conformationally uncoupled from the TATA box. Combining these results with proposed similarities between SNF2/SWI2 ATPases and helicases, we suggest that MOT1 uses ATP hydrolysis to translocate along the handle and thereby disrupt interactions between TBP and DNA.

Mot1 is an essential

Exact positions of 5-methylcytosine (m 5 C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which then convert to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed.