Effect of the non-conserved N-terminus on the DNA binding activity of the yeast TATA binding protein.
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.
Kuddus
R
R
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA 15261.
Schmidt
M C
MC
D001483:Base Sequence; D004271:DNA, Fungal / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012996:Solutions; D016385:TATA Box; D035181:TATA-Box Binding Protein; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D012996:Solutions; D035181:TATA-Box Binding Protein; D014157:Transcription Factors