Alanine scanning site-directed mutagenesis of the zinc fingers of transcription factor ADR1: residues that contact DNA and that transactivate.
To identify functionally important amino acids in the two zinc fingers of transcription factor ADR1 [alcohol dehydrogenase (ADH) II synthesis regulator], oligonucleotide-directed mutagenesis was used to substitute alanine for the original amino acid at each position in both fingers. The effects of these mutations on DNA binding and thermal stability of ADR1 in vitro and on activation of ADH2 expression in vivo were measured. The DNA binding activity was remarkably heatstable. Amino acids that are candidates for DNA contact sites were identified in the finger-tip and alpha-helical region of each finger, three in the first finger and two in the second. Unexpectedly, an acidic residue in the first finger was essential for transactivation, but its replacement by alanine had no effect on DNA binding. Substitution at several highly conserved positions did not affect ADR1 functions. The ADR1 zinc fingers appear to make relatively few energetically significant contacts to DNA, perhaps as few as three in the first finger and one in the second.
Department of Biochemistry SJ70, University of Washington, Seattle 98195.
S K
SK
Thukral
M L
ML
Morrison
E T
ET
Young
D000409:Alanine; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D016335:Zinc Fingers / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C074682:ADR1 protein, S cerevisiae; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D007527:Isoenzymes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D004247:DNA; D000426:Alcohol Dehydrogenase; D001616:beta-Galactosidase; D000409:Alanine