Structural dissection of the DNA-binding domain of the yeast transcriptional activator GAL4 reveals an alpha-helical region responsible for dimerization.
Limited proteolysis of the DNA-binding domain (residues 1-147) of the yeast transcriptional activator GAL4 has been used to define more precisely the subdomain structure required for DNA binding and dimerization. Two regions of the protein were found to be resistant to proteolysis: the cysteine-rich, zinc-binding region (residues 6-43) and a hydrophobic sequence between residues 52 and 97. Carboxy-terminal deletion fragments of the DNA-binding domain were generated and assayed by DNase 1 footprinting. This showed that the affinity of DNA binding depends on the sequence between residues 65 and 94. Structural comparisons by UV circular dichroism (CD) were made and the difference CD spectra indicate that strong alpha-helical content is found specifically in the region between residues 65 and 94, which previous studies have shown to enable dimerization and in this study the formation of a stable protein-DNA complex.
Gadhavi
P L
PL
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, United Kingdom. p-gadhav@nimr.mrc.ac.uk
D000595:Amino Acid Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D019281:Dimerization; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D029701:Saccharomyces cerevisiae Proteins; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C064255:GAL4 (1-147) protein, S cerevisiae; C052130:GAL4 protein, S cerevisiae; D010446:Peptide Fragments; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA