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DNA binding and bending by the transcription factors GAL4(62*) and GAL4(149*)
Author(s) -
Rodgers Karla K.,
Coleman Joseph E.
Publication year - 1994
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560030409
Subject(s) - dna , affinities , dimer , chemistry , dna binding domain , binding domain , binding site , stereochemistry , dna binding site , dna binding protein , transcription factor , biochemistry , promoter , gene , gene expression , organic chemistry
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, Zn 2 C 6 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 Zn 2 and Zn 1 forms of the N‐terminal 149 residues of GAL4, 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 affinities than Zn 2 GAL4(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, >10 3 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 Zn 2 GAL4(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 > 10 3 lower affinity of GAL4(62*) for the UAS G DNA can be accounted for by its failure to form dimers in solution. Zn 2 ‐, Zn 1 ‐, or Cd 2 GAL4(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.