Tight cooperation between Mot1p and NC2β in regulating genome-wide transcription, repression of transcription following heat shock induction and genetic interaction with SAGA

TATA-binding protein (TBP) is central to the regulation of eukaryotic transcription initiation. Recruitment of TBP to target genes can be positively regulated by one of two basal transcription factor complexes: SAGA or TFIID. Negative regulation of TBP promoter association can be performed by Mot1p or the NC2 complex. Recent evidence suggests that Mot1p, NC2 and TBP form a DNA-dependent protein complex. Here, we compare the functions of Mot1p and NC2βduring basal and activated transcription using the anchor-away technique for conditional nuclear depletion. Genome-wide expression analysis indicates that both proteins regulate a highly similar set of genes. Upregulated genes were enriched for SAGA occupancy, while downregulated genes preferred TFIID binding. Mot1p and NC2β depletion during heat shock resulted in failure to downregulate gene expression after initial activation, which was accompanied by increased TBP and RNA pol II promoter occupancies. Depletion of Mot1p or NC2β displayed preferential synthetic lethality with the TBP-interaction module of SAGA. Our results support the model that Mot1p and NC2β directly cooperate in vivo to regulate TBP function, and that they are involved in maintaining basal expression levels as well as in resetting gene expression after induction by stress

Analyzing the dynamics of single TBP-DNA-NC2 complexes using hidden markov models.

Single-pair Forster resonance energy transfer (spFRET) has become an important tool for investigating conformational dynamics in biological systems. To extract dynamic information from the spFRET traces measured with total internal reflection fluorescence microscopy, we extended the hidden Markov model (HMM) approach. In our extended HMM analysis, we incorporated the photon-shot noise from camera-based systems into the HMM. Thus, the variance in Forster resonance energy transfer (FRET) efficiency of the various states, which is typically a fitted parameter, is explicitly included in the analysis estimated from the number of detected photons. It is also possible to include an additional broadening of the FRET state, which would then only reflect the inherent flexibility of the dynamic biological systems. This approach is useful when comparing the dynamics of individual molecules for which the total intensities vary significantly. We used spFRET with the extended HMM analysis to investigate the dynamics of TATA-box-binding protein (TBP) on promoter DNA in the presence of negative cofactor 2 (NC2). We compared the dynamics of two promoters as well as DNAs of different length and labeling location. For the adenovirus major late promoter, four FRET states were observed; three states correspond to different conformations of the DNA in the TBP-DNA-NC2 complex and a four-state model in which the complex has shifted along the DNA. The HMM analysis revealed that the states are connected via a linear, four-well model. For the H2B promoter, more complex dynamics were observed. By clustering the FRET states detected with the HMM analysis, we could compare the general dynamics observed for the two promoter sequences. We observed that the dynamics from a stretched DNA conformation to a bent conformation for the two promoters were similar, whereas the bent conformation of the TBP-DNA-NC2 complex for the H2B promoter is approximately three times more stable than for the adenovirus major late promoter

NC2 mobilizes TBP on core promoter TATA boxes.

The general transcription factors (GTFs) of eukaryotic RNA polymerase II, in a process facilitated by regulatory and accessory factors, target promoters through synergistic interactions with core elements. The specific binding of the TATA box-binding protein (TBP) to the TATA box has led to the assumption that GTFs recognize promoters directly, producing a preinitiation complex at a defined position. Using biochemical analysis as well as biophysical single-pair Förster resonance energy transfer, we now provide evidence that negative cofactor-2 (NC2) induces dynamic conformational changes in the TBP-DNA complex that allow it to escape and return to TATA-binding mode. This can lead to movement of TBP along the DNA away from TATA