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

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 DNA ‘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 Can Activate Basal Transcription In Vitro by Regulating the Distribution of TATA Binding Protein between

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The NEF4 Complex Regulates Rad4 Levels and Utilizes Snf2/Swi2-Related ATPase Activity for Nucleotide Excision Repair

Nucleotide excision repair factor 4 (NEF4) is required for repair of nontranscribed DNA in Saccharomyces cerevisiae. Rad7 and the Snf2/Swi2-related ATPase Rad16 are NEF4 subunits. We report previously unrecognized similarity between Rad7 and F-box proteins. Rad16 contains a RING domain embedded within its ATPase domain, and the presence of these motifs in NEF4 suggested that NEF4 functions as both an ATPase and an E3 ubiquitin ligase. Mutational analysis provides strong support for this model. The Rad16 ATPase is important for NEF4 function in vivo, and genetic analysis uncovered new interactions between NEF4 and Rad23, a repair factor that links repair to proteasome function. Elc1 is the yeast homologue of a mammalian E3 subunit, and it is a novel component of NEF4. Moreover, the E2s Ubc9 and Ubc13 were linked to the NEF4 repair pathway by genetic criteria. Mutations in NEF4 or Ubc13 result in elevated levels of the DNA damage recognition protein Rad4 and an increase in ubiquitylated species of Rad23. As Rad23 also controls Rad4 levels, these results suggest a complex system for globally regulating repair activity in vivo by controlling turnover of Rad4

RNA synthesis precision is regulated by preinitiation complex turnover

TATA-binding protein (TBP) nucleates the assembly of the transcription preinitiation complex (PIC), and although TBP can bind promoters with high stability in vitro, recent results establish that virtually the entire TBP population is highly dynamic in yeast nuclei in vivo. This dynamic behavior is surprising in light of models that posit that a stable TBP-containing scaffold facilitates transcription reinitiation at active promoters. The dynamic behavior of TBP is a consequence of the enzymatic activity of the essential Snf2/Swi2 ATPase Mot1, suggesting that ensuring a highly mobile TBP population is critical for transcriptional regulation on a global scale. Here high-resolution tiling arrays were used to define how perturbed TBP dynamics impact the precision of RNA synthesis in Saccharomyces cerevisiae. We find that Mot1 plays a broad role in establishing the precision and efficiency of RNA synthesis: In mot1-42 cells, RNA length changes were observed for 713 genes, about twice the number observed in set2Δ cells, which display a previously reported propensity for spurious initiation within open reading frames. Loss of Mot1 led to both aberrant transcription initiation and termination, with prematurely terminated transcripts representing the largest class of events. Genetic and genomic analyses support the conclusion that these effects on RNA length are mechanistically tied to dynamic TBP occupancies at certain types of promoters. These results suggest a new model whereby dynamic disassembly of the PIC can influence productive RNA synthesis

Structural basis for recognition and remodeling of the TBP:DNA:NC2 complex by Mot1

Swi2/Snf2 ATPases remodel substrates such as nucleosomes and transcription complexes to control a wide range of DNA-associated processes, but detailed structural information on the ATP-dependent remodeling reactions is largely absent. The single subunit remodeler Mot1 (modifier of transcription 1) dissociates TATA box-binding protein (TBP):DNA complexes, offering a useful system to address the structural mechanisms of Swi2/Snf2 ATPases. Here, we report the crystal structure of the N-terminal domain of Mot1 in complex with TBP, DNA, and the transcription regulator negative cofactor 2 (NC2). Our data show that Mot1 reduces DNA:NC2 interactions and unbends DNA as compared to the TBP:DNA:NC2 state, suggesting that Mot1 primes TBP:NC2 displacement in an ATP-independent manner. Electron microscopy and cross-linking data suggest that the Swi2/Snf2 domain of Mot1 associates with the upstream DNA and the histone fold of NC2, thereby revealing parallels to some nucleosome remodelers. This study provides a structural framework for how a Swi2/Snf2 ATPase interacts with its substrate DNA:protein complex