Analysis of specific protein-DNA interactions by bacterial one-hybrid assay.

The DNA-binding specificity of transcription factors allows the prediction of regulatory targets in a genome. However, very few factor specificities have been characterized and still too little is known about how these proteins interact with their targets to make predictions a priori. To provide a greater understanding of how proteins and DNA interact, we have developed a bacterial one-hybrid system that allows the sensitive, high-throughput, and cost-effective assay of the interaction at the protein-DNA interface. This system makes survival of the bacteria dependent on activation of the reporter gene and therefore dependent on the protein-DNA interaction that recruits the polymerase. We have used this system to characterize DNA-binding specificities for representative members of the most common DNA-binding domain (DBD) families. We have also been able to engineer DBDs with novel specificity to be used as artificial transcription factors and zinc finger nucleases. The B1H assay provides a simple and inexpensive method to investigate protein-DNA interactions that is accessible to essentially any laboratory.