DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*)

Abstract

The DNA binding domain of the GAL4 transcription factor from yeast is located in the N-terminal 60 residues of the polypeptide of 881 amino acids. This domain binds 2 Zn ions, which form a binuclear cluster, Zn₂C₆ with 6 C residues, two of which bridge the 2 metal ions (Gardner KH et al., 1991, Biochemistry 30:11292-11302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recognize the specific DNA sequence, UAS_G, to which GAL4 binds as a dimer. Gel retardation assays have been utilized to determine the relative affinities of the Zn₂ and Zn₁ forms of the N-terminal 149 residues of GAL4, GAL4(149*), for UAS_G DNA sequences. We show that Cd₂- and Zn₁ GAL4(149*) bind to UAS_G DNA with 2-fold and 4-8-fold lower affinities than Zn₂GAL4(149*), respectively. Thus, the metal species and the number of metal ions bound have measurable effects on the specific DNA binding affinity of GAL4, but these differences are small in comparison to the ratio, >10³ under some conditions, that characterizes the specific to nonspecific DNA binding affinities of the N-terminal fragments of GAL4. A shorter N-terminal fragment, GAL4(62*), although it continues to recognize the UAS_G sequence with a high degree of specificity, binds with 1,000-2,000-fold lower affinity than does Zn₂GAL4(149*). Gel retardation titrations of a DNA containing 2 UAS_G sites with increasing concentrations of GAL4(62*) generate a series of 4 retarded bands in contrast to 2 retarded bands formed when the the same DNA is titrated with GAL4(149*). These data suggest that GAL4(62*) binds to the UAS_G sites as individual monomers that dimerize on the DNA, whereas GAL4(149*) binds the UAS_G DNA cooperatively as a dimer. The > 10³ lower affinity of GAL4(62*) for the UAS_G DNA can be accounted for by its failure to form dimers in solution. Zn₂-, Zn₁-, or Cd₂GAL4(149*) induces differential rates of gel migration in a series of circularly permutated UAS_G-containing DNA restriction fragments. Analysis of the data suggests that all 3 proteins cause a 26° angle of bend in the DNA when bound to 1 UAS_G site and 45° when bound to 2 tandem UAS_G sites. The same assay shows that GAL4(62*) does not induce significant bending of the UAS_G DNA sequences. Thus, the additional subdomains found in the larger polypeptide fragment, GAL4(149*), must exert an additional force on the DNA either through direct contacts with the DNA or indirectly through altered protein conformation.