Identification of Protein-DNA Interactions Using Enhanced Yeast One-Hybrid Assays and a Semiautomated Approach

Yeast one-hybrid assays are an in vitro gene-centered approach to map transcription factor-DNA interactions. Here we describe this method and adaptations to screen for interactions between plant transcriptional regulators and their targets. Of particular note, the use of yeast one-hybrid assays fills in an important gap in available methodologies. When one is interested in a specific biological process of interest, the yeast one-hybrid assay is the only method that allows researchers to identify upstream regulators of the biological process of interest. This technique can be also used to further validate physical protein-DNA interactions or as a hypothesis-generating tool. In this method, promoters or DNA regions of interest are cloned and transformed into yeast and tested for interaction against a collection of transcription factors (TFs). Yeast one-hybrid screens are adaptable to the question the researcher is asking and the tools and components available. In this chapter we will describe large-scale and high-throughput Y1H screening; however, this can easily be scaled down for smaller studies.