Part of the Methods in Molecular Biology book series (MIMB, volume 177)
One-Hybrid Systems f hor Detecting Protein-DNA Interactions
The yeast two-hybrid assay has proven useful for detecting protein-protein interactions. A variation on the theme can be used for finding proteins that interact with a particular DNA sequence. The one-hybrid assay, so far carried out only in Saccharomyces cerevisiae, in its simplest form (Fig. 1) consists of a DNA sequence of interest placed upstream of a reporter gene. The reporter gene can be either on a plasmid (1) or integrated into the chromosome (2). The protein or library being tested is cloned into a vector that expresses that protein fused to a transcription activation domain (TAD), the equivalent of the prey protein in a two-hybrid assay. This hybrid protein is expressed in the strain carrying the reporter gene. If the protein is able to interact with the sequence of interest, by either binding directly to the DNA or indirectly via interaction with a DNA-binding protein, transcription of the reporter gene is activated.
KeywordsCysteine Proline Lysine Arginine Tryptophan
- 14.Liu, Q., Kasuga, M., Sakuma, Y., Abe, H., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1998) Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought-and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell 10, 1391–1406.PubMedCrossRefGoogle Scholar
© Humana Press Inc. 2001