Genetic approaches for dissecting protein-protein interactions

Many cellular processes require specific interactions between two different proteins. Two classical genetic approaches for identifying protein-protein interactions are the isolation of allele specific suppressors or mutations with synthetic lethal phenotypes. More recently a variety of gene fusion vectors have been developed for studying protein-protein interactions. This approach involves the fusion of potential protein-protein interaction domains of the protein of interest with the DNA-binding domain of an easily assayable reporter gene -- for example, the lambda repressor, the alpha subunit of E. coli RNA polymerase, the Bordetella pertussis cya protein, or the yeast Gal4 activator.

Lambda cI hybrids. Jim Hu and colleagues developed a two-hybrid system based upon fusions with the lambda repressor in E.coli (for a review see The system involves construction of gene fusions that result in the fusion of a protein to the N-terminus of the Lambda cI protein as shown in the diagram below. The Lambda cI protein must dimerize to bind to its operator site and repress transcription (A). The N-terminus of the Lambda cI protein retains the DNA binding domain but lacks the dimerization, so it cannot repress transcription (B). If another protein fused to the N-terminus the Lambda cI protein can dimerize, it will substitute for the missing dimerization domain of the Lambda cI protein (C). (More information on this system can be found on the Hu lab web site.)

Lambda cI - alpha subunit hybrids. Hochschild and colleagues developed another two-hybrid system that is based upon fusions with both the lambda repressor and the alpha subunit of RNA polymerase in E. coli. A gene of interest (the bait) is fused to the full-length bacteriophage lambda cI gene, resulting in a hybrid protein with the amino-terminal DNA-binding domain of lambda cI and the carboxyl-terminal dimerization domain of the bait protein. The cognate target gene is fused to a gene encoding the N-terminal domain of the -subunit of RNA polymerase. The bait is tethered to the operator sequence upstream of the reporter promoter through the DNA-binding domain of lambda cI. When the bait and target interact, they recruit and stabilize the binding of RNA polymerase close to the promoter and activate the transcription of the downstream reporter genes, lacZ and amp^R. (This system is marketed by Stratagene as the BacterioMatch Two-Hybrid System.)

Yeast two-hybrid. The yeast two-hybrid system is a sensitive assay for the detection of specific protein-protein interactions in Saccharomyces cerviasae. This system is based on the modular nature of certain eukaryotic transcriptional activators, which allow them to be divided into two separate functional domains: a DNA-binding domain which recognizes a specific DNA-binding site, and an activation domain which turns-on transcription of a reporter gene. The basic two-hybrid system uses the yeast GAL4 transcriptional activator which is required for expression of genes encoding proteins involved in galactose metabolism. However, there are now many other variations of the basic yeast two-hybrid system. (Many companies sell variations of the yeast two-hybrid systems. Several biotech companies market two-hybrid kits.


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Last modified November 6, 2003