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 Dros ophila melanogaster protein, Bicoid, to stimulate transcription of target g enes in precise sub-domains within the embryo. As a first step toward testi ng this idea, we devised a genetic screen to isolate mutations in Bicoid th at specifically disrupt cooperative interactions, but do not disrupt DNA re cognition or transcription activation. The screen was carried out in Saccha romyces cerevisiae and 12 cooperativity mutants were identified. The mutati ons 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 compar able to that of wild-type, indicating that DNA binding per se is not affect ed. However, these mutants failed to show cooperative coupling between high and low-affinity sites, and showed reduced activation of a reporter gene c arrying a natural Drosophila enhancer. Homology modeling indicated that non e of the four mutations is in residues that contact DNA. Instead, these res idues are likely to interact with other DNA-bound Bicoid monomers or other parts of the Bicoid protein. In vitro, the isolated homeodomains did not sh ow 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. (C) 2001 Academic Press.