We developed a simple and accurate method to define the sequence recognition properties of DNA-binding proteins. The method employs polymerase chain reaction (PCR) amplification of sequences selected from a mixture of random oligonucleotides by the gel mobility-shift assay. We used this method to define the sequence requirement of the binding domain of the yeast transcriptional activator GCN4. Using a total of 200 ng of purified protein and four cycles of binding and subsequent amplification, we identified the TGA-(C/G)TCA sequence as the binding consensus of GCN4, which is consistent with the previously reported recognition sequence. In addition, our data indicate that GCN4 can bind with lower affinity to sequences that differ from the optimal sequence in one or even two positions. The most common variation was the C to A at position +2. The majority of the substitutions that still allowed binding were 3' to the central C residue indicating that the two sides of the palindromic recognition sequence are not equivalent.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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