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-terminal60 residues of the polypeptide of 881 amino acids. This domain binds 2 Zn ions, which form a binuclear cluster, ZnzC,, with 6 C residues, two of which bridge the 2 metal ions (Gardner KH et al., 1991, Biochemistry 3 0 11292-1 1302). Binding of Zn or Cd to GAL4 induces the conformation of the protein necessary to recognize the specific DNA sequence, UASG, to which GAL4 binds as a dimer. Gel retardation assays have been utilized to determine the relative affinities of the Zn, and Zn, forms of the N-terminal 149 residues of GAL4, GAL4(149*), for UAS, DNA sequences. We show that Cd2-and ZnlGAL4(149*) bind to UASG DNA with 2-fold and 4-8-fold lower affinities than ZnzGAL4(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, >lo3 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 UASG sequence with a high degree of specificity, binds with 1,000-2,000-fold lower affinity than does ZnzGAL4(149*). Gel retardation titrations of a DNA containing 2 UASG 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 UASG sites as individual monomers that dimerize on the DNA, whereas GAL4(149*) binds the UASG DNA cooperatively as a dimer. The =IO3 lower affinity of GAL4(62*) for the UASG DNA can be accounted for by its failure to form dimers in solution. Zn,-, Znl-, or Cd2GAL4(149*) induces differential rates of gel migration in a series of circularly permutated UASGcontaining 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 UASG site and 45" when bound to 2 tandem UASG sites. The same assay shows that GAL4(62*) does not induce significant bending of the UAS, 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.

Coordination dynamics of biological zinc “clusters” in metallothioneins and in the DNA-binding domain of the transcription factor Gal4

“…Zinc exchange rates observed here are between 1 and 2 orders of magnitude faster than those reported for the Gal4(62*) protein, a shorter fragment, with free zinc ions (24). In this study, an examination revealed biphasic kinetics, with t 1/2 ϭ 24 h and t 1/2 ϭ 96 h at 4ЊC and pH 7.0 for both processes.…”

“…Preparation of Gal4. The storage buffer (10 mM Tris⅐Cl͞150 mM sodium chloride͞1 mM 2-mercaptoethanol͞10 M Zn 2ϩ , pH 8) of the bacterially expressed Gal4(149*) fragment (24) was exchanged against 10 mM Tris⅐Cl͞15 mM sodium chloride, pH 8.6, using Centricon-10 centrifugal concentrators (Amicon). At least 15 mM sodium chloride is necessary to solubilize the protein fragment at pH 8.6.…”

“…At least 15 mM sodium chloride is necessary to solubilize the protein fragment at pH 8.6. The metal content of the protein was 2.3 mol of zinc per monomer (M r ϭ 17,880) at a protein concentration of 20 M. Stock solutions of the protein were prepared at this concentration, because zinc analyses of Ͻ2 mol of zinc per monomer were measured at lower protein concentrations due to weaker binding and loss of one of the zinc atoms during equilibrium dialysis (24). Protein concentration was determined spectrophotometrically ( 280 ϭ 7300 M Ϫ1 ⅐cm Ϫ1 ) and confirmed by amino acid analysis.…”

A Zinc-binding Domain Involved in the Dimerization of RAG1

“…The DNA fragments were 32 P-labeled with [g-32 P]ATP using phage T4 polynucleotide kinase. Gel mobility shift assays were done as described with the exception that poly(dIdC) was used as the competitor DNA (Rodgers & Coleman, 1994).…”

Delivery of Intact Transcription Factor by Using Self‐Assembled Supramolecular Nanoparticles

“…As shown in Figure 1, three types of molecular recognition mechanisms were employed to facilitate the preparation of TF-encapsulated SNP (TF·DNA⊂SNPs). First, the specific binding (the dissociate constant K d ≈ 10 nM) [15] between GAL4-VP16 (TF) and pG5E4T-Fluc (DNA) led to the formation of an anionic TF·DNA complex. Second, the Ad/CD-based molecular recognition ( K =1.1 × 10 5 M −1 ) [16] was utilized to form the SNP vectors with cationic hydrogel cores.…”