Isolation of mutations that disrupt cooperative DNA binding by the Drosophila bicoid protein.
Cooperative DNA binding is thought to contribute to the ability of the Drosophila melanogaster protein, Bicoid, to stimulate transcription of target genes in precise sub-domains within the embryo. As a first step toward testing this idea, we devised a genetic screen to isolate mutations in Bicoid that specifically disrupt cooperative interactions, but do not disrupt DNA recognition or transcription activation. The screen was carried out in Saccharomyces cerevisiae and 12 cooperativity mutants were identified. The mutations map across most of the Bicoid protein, with some located within the DNA-binding domain (homeodomain). Four homeodomain mutants were characterized in yeast and shown to activate a single-site reporter gene to levels comparable to that of wild-type, indicating that DNA binding per se is not affected. However, these mutants failed to show cooperative coupling between high and low-affinity sites, and showed reduced activation of a reporter gene carrying a natural Drosophila enhancer. Homology modeling indicated that none of the four mutations is in residues that contact DNA. Instead, these residues are likely to interact with other DNA-bound Bicoid monomers or other parts of the Bicoid protein. In vitro, the isolated homeodomains did not show strong cooperativity defects, supporting the idea that other regions of Bicoid are also important for cooperativity. This study describes the first systematic screen to identify cooperativity mutations in a eukaryotic DNA-binding protein.
Burz
D S
DS
Molecular Genetics Program Wadsworth Center, New York State Department of Health, USA.
Hanes
S D
SD
D000494:Allosteric Regulation / Q000235:genetics; D000495:Allosteric Site; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000196:embryology / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000235:genetics; D018507:Gene Expression Regulation, Developmental; D017344:Genes, Insect / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D018398:Homeodomain Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics; D015533:Transcriptional Activation
D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D019476:Insect Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C056478:bcd protein, Drosophila; C061245:hb protein, Drosophila; D004247:DNA