Effect of the non-conserved N-terminus on the DNA binding activity of the yeast TATA binding protein

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Abstract
We have studied the DNA binding activity of recombinant yeast TATA Binding Protein (TBP) with particular interest in the role played by the non-conserved N-termlnal domain. By comparing the DNA binding activity of wild type yeast TBP with a mutant form of TBP that lacks the non-conserved N-termlnal domain (TBPΔ57), we have determined that the N-terminus of TBP alters both the shape and the stability of the TBP—DNA complex. Measurements of the DNA bending angle Indicate that the N-termlnus enhances the bending of the DNA that Is induced by TBP binding and greatly destabilizes the TBP—DNA complex during native gel electrophoresls. In solution, the N-termlnus has only a slight effect on the equilibrium dissociation constant and the dissociation rate constant. However, the N-termlnal domain reduces the association rate constant In a temperature dependent manner and Increases the apparent activation energy of the TBP—DNA complex formation by 3 kcal/mole. These data suggest that a conformational change involving the N-termlnus of TBP may be one of the Isomerizatlon steps In the formation of a stable TBP—DNA complex.