Mutational analysis of the GAL4-encoded transcriptional activator protein of Saccharomyces cerevisiae.
The GAL4 protein of Saccharomyces cerevisiae binds to DNA upstream of each of six genes and stimulates their transcription. To locate regions of the protein responsible for these processes, we identified and characterized 88 gal4 mutations selected in vivo to reduce the ability to GAL4 protein to activate transcription. These mutations alter two regions of GAL4 protein: the DNA binding domain, and the transcription activation domain. Some mutations in the DNA binding domain that abolish the ability of GAL4 protein to bind to DNA in vitro change amino acid residues proposed to form a zinc finger, confirming that this structure is indeed involved in DNA binding. Four different amino acid changes in the zinc finger appear to reduce (but not abolish) the affinity of GAL4 protein for zinc ions, thereby identifying some of the amino acids involved in forming the zinc-binding structure. Several other mutations that abolish the DNA binding activity of the protein alter the 20 amino acids adjacent to the zinc finger, suggesting that these residues are part of the DNA binding domain. Two amino acid changes in the region adjacent to the zinc finger also appear to affect the ability of GAL4 protein to bind zinc ions, suggesting that this region of the protein can influence the structure of the zinc binding domain. The transcription activation domain of GAL4 protein is remarkably resistant to single amino acid changes: only 4 of the 42 mutations that alter this region of the protein are of the missense type. This observation is consistent with other lines of evidence that GAL4 protein possesses multiple transcription activation domains with unusual sequence flexibility.
Johnston
M
M
Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 63110.
Dover
J
J
D000483:Alleles; D000595:Amino Acid Sequence; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005796:Genes; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D013045:Species Specificity; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors