EFFECT OF THE NONCONSERVED N-TERMINUS ON THE DNA-BINDING ACTIVITY OF THE YEAST TATA-BINDING PROTEIN

We have studied the DNA binding activity of recombinant yeast TATA Bin ding Protein (TBP) with particular interest in the role played by the non-conserved N-terminal domain. By comparing the DNA binding activity of wild type yeast TBP with a mutant form of TBP that lacks the non-c onserved N-terminal domain (TBPA57), we have determined that the N-ter minus of TBP alters both the shape and the stability of the TBP - DNA complex. Measurements of the DNA bending angle indicate that the N-ter minus enhances the bending of the DNA that is induced by TBP binding a nd greatly destabilizes the TBP - DNA complex during native gel electr ophoresis. In solution, the N-terminus has only a slight effect on the equilibrium dissociation constant and the dissociation rate constant. However, the N-terminal domain reduces the association rate constant in a temperature dependent manner and increases the apparent activatio n energy of the TBP - DNA complex formation by 3 kcal/mole. These data suggest that a conformational change involving the N-terminus of TBP may be one of the isomerization steps in the formation of a stable TBP - DNA complex.