DNA-BINDING AND BENDING BY THE TRANSCRIPTION FACTORS GAL4(62-ASTERISK) AND GAL4(149-ASTERISK)

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, Zn (2)C6(,) with 6 C residues, two of which bridge the 2 metal ions (Gard ner KH et al., 1991, Biochemistry 30:11292-11302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recogniz e the specific DNA sequence, UAS(G), to which GAL4 binds as a dimer. G el retardation assays have been utilized to determine the relative aff inities of the Zn-2 and Zn-1 forms of the N-terminal 149 residues of G AL4, GAL4(149()), for UAS(G) DNA sequences. We show that Cd-2- and Zn (1)GAL4(149()) bind to UAS(G) DNA with 2-fold and 4-8-fold lower affi nities than Zn(2)GAL4(149()), respectively. Thus, the metal species a nd the number of metal ions bound have measurable effects on the speci fic DNA binding affinity of GAL4, but these differences are small in c omparison to the ratio, >10(3) under some conditions, that characteriz es the specific to nonspecific DNA binding affinities of the N-termina l fragments of GAL4. A shorter N-terminal fragment, GAL4(62()), altho ugh it continues to recognize the UAS(G) sequence with a high degree o f specificity, binds with 1,000-2,000-fold lower affinity than does Zn (2)GAL4(149()). Gel retardation titrations of a DNA containing 2 UAS( G) sites with increasing concentrations of GAL4(62()) generate a seri es 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 G AL4(62()) binds to the UAS(G) sites as individual monomers that dimer ize on the DNA, whereas GAL4(149()) binds the UAS(G) DNA cooperativel y as a dimer. The approximate to 10(3) lower affinity of GAL4(62()) f or the UAS(G) DNA can be accounted for by its failure to form dimers i n solution. Zn-2-, Zn-1-, or Cd(2)GAL4(149()) induces differential ra tes of gel migration in a series of circularly permutated UAS(G)-conta ining DNA restriction fragments. Analysis of the data suggests that al l 3 proteins cause a 26 degrees angle of bend in the DNA when bound to 1 UAS(G) site and 45 degrees 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 addition al force on the DNA either through direct contacts with the DNA or ind irectly through altered protein conformation.