Analysis of Specific Protein–DNA Interactions by Bacterial One-Hybrid Assay

Abstract

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.