Title: Unraveling the Dynamics of TBP-NC2 with Hidden Markov Modeling
Abstract: One of the early steps in initiation of transcription is the binding of the TATA box Binding Protein (TBP) to a core-promoter TATA Box. After binding, the transcription complex is assembled in cooperation with our general transcription factors. The binding of TBP to DNA is also a prime target for regulating gene transcription and many cofactors can interact with TBP at this initial stage. In a recent publication (Schluesche et al), we showed that the binding of the Negative Cofactor 2 (NC2) leads to dynamic behavior of the TBP-NC2 complex along the DNA using single-pair Förster Resonance Energy Transfer (spFRET). To extract detailed kinetic information from the single molecule experiments, we have adapted a Hidden Markov Model to analyze the spFRET data collected using a EMCCD camera. With add of the HMM analysis, it is possible to determine how many states are available which transitions are possible, providing new insights into the origins of these different states. We performed measurements on four DNA strands, two containing the AdML promoter with identical labeling but different DNA lengths and two containing the H2B promoter with labels at two different positions on the DNA. Results of the AdML promoter showed the high reproducibility of the analysis method. Four FRET states were clearly observable. For the H2B promoter, many more states were observable in the HMM analysis showing a high complexity of dynamics but yet very similar patterns as observed for the AdML promoter. Schluesche et al 2007 NSMB 14:1196-1201