|M.Sc Thesis||Department of Biology|
The TATA-box binding protein (TBP) is one of the most important proteins involved in transcriptional regulation in all eukaryotes. TBP recognition of its target sites (TATA-boxes) is the first step in building the preinitiation complex. The recognition between TBP and TATA-boxes involves structural recognition of DNA conformation, termed “indirect readout”. In order to explore the mechanism of indirect readout between DNA and proteins, I studied the TBP/TATA-box system as a model system.
In the first part of my research I determined the influence of sequences flanking different TATA-boxes on TBP/TATA-box interaction. In this part of the work I found that the influence of the flanking sequences may depend on the structural properties of the TATA-box: when TATA-box have a dominant and invariable structure, such as that observed in A-tracts, the flanking sequences have very little effect on the complex stability and binding affinity in TBP/TATA-box interaction. On the other hand, when the TATA-box has a flexible and context-dependent structure, such as observed in alternating (T-A)n runs, the flanking sequences have significant effect on TBP/TATA-box interaction. By changing the flanking sequences alone we managed to greatly improve the complex stability and binding affinity between DNA and TBP in the case of alternating (T-A)n TATA-boxes.
In the second part of this work I studied how alteration within TATA-box itself influences the interaction between TBP and TATA-boxes. Experiments in which we determined the binding affinity of TBP caused by TBP-induced TATA-box bending to all consensus-like TATA-boxes resulted in a division of the studied sequences to two groups. The first group includes sequences with more invariable conformation because of a central A-A step. In the second group a central A-T step made the sequences more structurally flexible. In each group there are different correlations between the TATA-box sequence, its structural properties and the characteristics of its interaction with TBP.
In the third part of this work I established a high-throughput method for studying the dynamic flexibility of DNA molecules. In this method the flexibility of DNA molecules in solution is monitored using energy transfer between two fluorescent molecules attached to the two ends of a studied DNA sequence. Data obtained by this method allows calculating different aspects of DNA structure and flexibility when the experiments are compared to simulations of DNA models.