Spectroscopic studies of the DNA binding site of the GAL4 "zinc finger" protein.
The yeast GAL4 protein, a transcriptional activator of genes involved in galactose metabolism, binds as a dimer to several closely related seventeen base pair upstream activation sequences (UASGs) that are nearly symmetric about a central dT-dA base pair. A previous study of a GAL4-UASG complex (Carey, M., Kakidani, H., Leatherwood, J., Mostashari, F. and Ptashne, M. (1989) J. Mol. Biol. 209, 423-432) elucidated a pattern of contacts consistent with the protein partially wrapping itself around the helical cylinder, assuming a B-form conformation for the DNA. Alternatively, both monomers could sit on one face of the cylinder if the DNA exists in an underwound conformation such as A-form. Spectroscopic studies that distinguish between these models are reported here. Oligonucleotides containing the consensus UASG or a nine base pair "half site" both exhibit circular dichroism (CD) spectra characteristic of B-form DNA. Two-dimensional NMR studies of the half-site also indicate a B-form conformation. When a GAL4 protein fragment containing the entire DNA-binding and dimerization domains (amino acids 1-140) is bound to the UASG, the CD spectrum above 240 nm changes only slightly, and not in a manner consistent with DNA unwinding. Our studies suggest that the UASG does not adopt an unusual underwound conformation in the absence or presence of the GAL4 protein, and favor the model in which the dimer partially wraps around the helix cylinder.
Hansen
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A
Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Van Hoy
M
M
Kodadek
T
T
D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D005656:Fungal Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry*; D011485:Protein Binding; D012045:Regulatory Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D009841:Oligonucleotides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors