Characterization of the DNA-binding Domains From the Yeast Cell-Cycle Transcription Factors Mbp1 and Swi4

Biochemistry. 2000 Apr 11;39(14):3943-54. doi: 10.1021/bi992212i.

Abstract

The minimal DNA-binding domains of the Saccharomyces cerevisiae transcription factors Mbp1 and Swi4 have been identified and their DNA binding properties have been investigated by a combination of methods. An approximately 100 residue region of sequence homology at the N-termini of Mbp1 and Swi4 is necessary but not sufficient for full DNA binding activity. Unexpectedly, nonconserved residues C-terminal to the core domain are essential for DNA binding. Proteolysis of Mbp1 and Swi4 DNA-protein complexes has revealed the extent of these sequences, and C-terminally extended molecules with substantially enhanced DNA binding activity compared to the core domains alone have been produced. The extended Mbp1 and Swi4 proteins bind to their cognate sites with similar affinity [K(A) approximately (1-4) x 10(6) M(-)(1)] and with a 1:1 stoichiometry. However, alanine substitution of two lysine residues (116 and 122) within the C-terminal extension (tail) of Mbp1 considerably reduces the apparent affinity for an MCB (MluI cell-cycle box) containing oligonucleotide. Both Mbp1 and Swi4 are specific for their cognate sites with respect to nonspecific DNA but exhibit similar affinities for the SCB (Swi4/Swi6 cell-cycle box) and MCB consensus elements. Circular dichroism and (1)H NMR spectroscopy reveal that complex formation results in substantial perturbations of base stacking interactions upon DNA binding. These are localized to a central 5'-d(C-A/G-CG)-3' region common to both MCB and SCB sequences consistent with the observed pattern of specificity. Changes in the backbone amide proton and nitrogen chemical shifts upon DNA binding have enabled us to experimentally define a DNA-binding surface on the core N-terminal domain of Mbp1 that is associated with a putative winged helix-turn-helix motif. Furthermore, significant chemical shift differences occur within the C-terminal tail of Mbp1, supporting the notion of two structurally distinct DNA-binding regions within these proteins.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Binding Sites
  • Cell Cycle
  • DNA / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Fungal Proteins / chemistry*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism
  • Molecular Sequence Data
  • Saccharomyces cerevisiae / cytology
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism*
  • Saccharomyces cerevisiae Proteins*
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • DNA-Binding Proteins
  • Fungal Proteins
  • MBP1 protein, S cerevisiae
  • SWI4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • DNA