Structure-function analysis of the DNA binding domain of Saccharomyces cerevisiae ABF1.
To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496.
Cho
G
G
Department of Biology Education, Seoul National University, Korea.
Kim
J
J
Rho
H M
HM
Jung
G
G
D000595:Amino Acid Sequence; D001483:Base Sequence; D004271:DNA, Fungal / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005800:Genes, Fungal / Q000235:genetics; D018257:Helix-Loop-Helix Motifs / Q000235:genetics; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D016335:Zinc Fingers / Q000235:genetics
C061886:ABF1 protein, S cerevisiae; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors