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-terminal domain. By comparing the DNA binding activity of wild type yeast TBP with a mutant form of TBP that lacks the non-conserved N-terminal domain (TBP delta 57), 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-terminus enhances the bending of the DNA that is induced by TBP binding and greatly destabilizes the TBP-DNA complex during native gel electrophoresis. 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 activation 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.

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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

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Kuddus R, Schmidt MC

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