TATA Binding Protein (TBP) has important functions in transcription initiation by all three eukaryotic nuclear RNA polymerases. In case of RNA polymerase II (pol II), the enzyme responsible for transcribing protein-encoding genes, TBP is the first of the general transcription factors to bind to the promoter. We have studied the interactions of yeast TBP (yTBP) with DNA sequences dereived from the Adenovirus 2 Major Late (ML) promoter, the mouse ß-globin promoter, and with long DNA co-polymers.

Here, I present chemical interference studies of TBP on the mouse ß-globin promoter. These studies revealed that the interactions between TBP and the mouse ß-globin promoter are very similar to those between TBP and the ML promoter. I further showed that at least for the ß-globin promoter the integrity of the sugar-phosphate backbone is essential for binding of TBP. In contrast, disruption of base stacking interactions had no deleterious effect. At specific locations within the TATA box disrupted base stacking enhanced TBP binding.

Changes in the intrinsic fluorescence emission spectrum of yTBP upon binding to DNA were used to examine several properties of binding. With this technique, we were able to distinguish yTPB bound specifically to TATA boxes from yTBP bound at other sequences. The specific binding of TBP to a TATA box was entropy driven, with most of the binding free energy coming from the release of condensed counterions. A mutation in the TATA box affected all binding parameters.

We observed individual TBP-DNA complexes by scanning force microscopy. These studies clearly demonstrated that TBP bends the DNA upon binding. We were able to visualize both TBP bound specifically at the TATA box and TBP complexes formed at other DNA sequences. These types of complexes differed in their bending properties.