Two Hybrid Analysis

The two-hybrid method was conceived and developed by Stan Fields and coworkers as a technique to detect binding (physical interaction) between two proteins under in vivo conditions (Fields & Song, 1989). The concept underlying the two-hybrid method is based on the modular structure of some transcription activators and on the use of reporter genes. The reader should be sure to carefully review these subjects before attempting to understand the two-hybrid method.


Stan Fields conceived of the idea for two-hybrid analysis based on studies of transcription activators that indicated that the DNA-binding region and the region required for transcription activation consist of distinct domains of the protein that one could mix and match. He surmised that these domains may not even have to be part of the same protein. Instead, maybe they only have to interact with one another. If this interaction is strong enough to be capable of tethering the activation domain to the DNA-binding domain, then this might be enough for the complex to function as a transcription activator. So he tried it out with two proteins that he knew bound to one another. Let us call them proteins X and Y.

Two fusion genes are constructed using the genes encoding proteins X (gene X) and protein Y (gene Y) as follows. Gene X is fused, in-frame to a short sequence encoding the DNA-binding domain of either lexA repressor (residues 1 -87) or Gal4 activator (residues 1-147) creating a hybrid gene encoding a LexABD-protein X fusion protein (see Figure 10.1) or Gal4BD-protein X. Gene Y is fused, in-frame to GAL4 codons 768-881 (containing the Gal4p transactivation domain) creating a hybrid gene encoding a Gal4AD-protein Y fusion protein. Both fusion gene constructions are expressed from high-level constitutive promoters so that the proteins are abundantly made under commonly used growth conditions. The fusion containing the DNA-binding domain is often referred to as the 'bait' and the fusion containing the activation domain is referred to as the 'prey'. Both fusion gene constructions are carried on plasmid vectors with different nutritional selection genes. This allows both constructions to be maintained together in the same yeast host strain. Two-hybrid kits are commercially available that contain bait and prey plasmids with a multiple cloning site positioned to facilitate construction of the in-frame fusions.

To assay protein X-protein Y interaction, both hybrid fusion genes are expressed in the same yeast host cell that also contains a reporter gene appropriate to the bait construction. That is, the promoter of the reporter gene contains the DNA sequence to which the bait fusion protein binds. If the bait fusion uses the LexABD, the reporter must contain the short LexA operator sequence, which is often in several tandem copies to ensure strong binding of the bait to the reporter. Similarly, if the bait fusion uses the Gal4BD, the reporter must contain the Gal4p DNA-binding site.

LexA DNA-binding domain-protein X fusion (bait fusion)

LexA DNA-binding domain

Protein X

Gal4p transactivation domain-protein Y fusion (prey fusion)

Gal4 activation domain

Protein Y

Figure 10.1 Construction of bait and prey fusions

Figure 10.1 Construction of bait and prey fusions

LexA operator site

Activation of transcription lacZ

LexA operator site

Figure 10.2 The two-hybrid interaction

As shown in Figure 10.2, the bait fusion protein will bind to its binding site in the promoter of the reporter gene. If proteins X and Y bind to one another, then the interaction will bring the activation domain of the Gal4p-protein Y fusion protein, the prey, into proximity to the transcription start site of the reporter gene and activate the transcription of the reporter gene. Assaying the level of expression of the reporter gene product monitors the rate of transcription of the reporter gene and provides a semi-quantitative evaluation of the strength of the binding between proteins X and Y.

The two-hybrid method can be used to demonstrate whether two proteins interact and under what conditions they interact. One can also test whether mutations in either protein affect the interaction and thereby identify the specific residues involved in the interaction. By using only short sections of the proteins in the hybrid constructions, one can also map the portion of each protein involved in the interaction.

The two-hybrid method can also be used to screen a library for novel proteins that interact with the bait fusion protein. (Of course, the bait fusion protein cannot itself have activation activity.) To make the prey library, random genomic DNA fragments (or cDNA fragments) are fused to the Gal4 activation domain using the prey vector. The library is then transformed into a yeast host cell containing the reporter gene and the bait fusion gene. Transformants are selected and then all are screened for expression of the reporter gene. If the reporter gene is lacZ, one can screen for blue colonies on X-gal plates.

Initial characterization of the positive transformants must be carried out to eliminate 'false positives'. Much of the literature on two-hybrid analysis discusses false positives and other pitfalls of the method. An example of a false positive is a prey fusion protein that binds, either directly or indirectly, to the promoter region of the reporter gene. Before undertaking a two-hybrid screen it would be very valuable to thoroughly read this literature. After the researcher is convinced that the interaction detected by the two-hybrid method is biologically relevant, the prey plasmid is recovered and the insert fragment sequenced and characterized.

The interaction detected by a positive two-hybrid result does not necessarily indicate that the two proteins being studied truly interact directly. It is possible that they both interact with the same protein as part of a multiprotein complex and a third protein might be needed to bridge the two fusion proteins thereby holding them together. To demonstrate direct interaction, one could use genetic approaches such as allele-specific suppression or enhancement or biochemical approaches such as coprecipitation. Other pitfalls to the proper interpretation of the significance of the interaction exist, particularly for researchers using the yeast two-hybrid system to study proteins from other organisms. Two proteins might be capable of interacting under the conditions of the yeast two-hybrid assay (abundant expression and localization to the host cell nucleus) but not under normal conditions. The two proteins in their normal host cell may be found in different compartments or expressed in different cell types. Therefore, in these situations the interaction in yeast is an artifact. A positive two-hybrid result is just the beginning and a great deal more genetic and biochemical analysis must be done to demonstrate the biological significance of the result.

Several variations of the basic two-hybrid method have been developed to improve the efficiency of introducing the library plasmid into the host tester strain and the detection of an interaction. This is particularly important when screening a large library such as a mammalian cDNA library. HIS3 or other nutritional genes can be used instead of lacZ as the reporter gene. In this way one can select for the expression of the nutritional gene rather than screen for the expression of lacZ. HIS3 is especially good for detecting weak interactions because only very low levels of HIS3 expression are needed for the His+ phenotype. The most significant modification to the two-hybrid method was conceived of by Bendixen et al. (1994) and Brent & Finley (1994). Instead of using transformation to introduce the 'prey' plasmid library into cells carrying the 'bait' plasmid, they used a/a mating between a large pool of cells transformed with the 'prey' library plasmids and cells carrying the 'bait' plasmid to construct a double transformant containing both plasmids.


Variations of the two-hybrid analysis method have been developed to explore DNA-protein and RNA protein interactions.

One-hybrid analysis is used to identify proteins that interact with a specific DNA sequence. This sequence is inserted into the promoter of a reporter gene: for example, in place of the LexA operator site of the lacZ reporter shown in Figure 10.2. A fusion gene library similar to the 'prey' library described above that fuses the library proteins to a transcription activation domain is introduced into a host strain carrying the reporter. Any hybrid proteins capable of binding to the DNA sequence of interest will activate the reporter gene expression.

Three-hybrid analysis was developed to study the RNA-protein interaction (reviewed in Kraemer et al., 2000). The third hybrid construct in this method is an RNA hybrid molecule. The bait hybrid protein binds one portion of this hybrid RNA. The other portion of the hybrid RNA is the sequence of interest. Fusion proteins of the 'prey' library are screened for their ability to bind to this RNA sequence of interest that is tethered to the reporter gene promoter via the bait hybrid protein.


Bartel, P.L. & S. Fields (1995) Analyzing protein-protein interactions using two-hybrid system. Meth. Enzymol. 254: 241-263. Bendixen, C., S. Gangloff, & R. Rothstein (1994) A yeast mating-selection scheme for detection of protein-protein interactions. Nucleic Acids Res. 11: 1778-1779. Brent, R. & R.L. Finley (1994) Interaction mating reveals binary and ternary connections between Drosophila cell cycle regulators. Proc. Natl Acad. Sci. USA 91: 12980-12984. Brent, R. & R.L. Finley (1997) Understanding gene and allele function with two-hybrid method. Ann. Rev. Genet. 31: 663-704. Chien, C.T., P.L. Bartel, R. Sternglanz, & S. Fields (1991) The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc. Natl Acad. Sci. USA 88: 9578-9582. Fields, S. & Song (1989) A novel genetic system to detect protein-protein interactions. Nature 340: 245-246.

Fields, S. & R. Sternglanz (1994) The two-hybrid system: an assay for protein-protein interaction. Trends Genet. 10: 286-292. James, P., J. Halladay, & E.A. Craig (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144: 1425-1436. Kraemer, B., B. Zhang, D. SenGupta, S. Fields, & M. Wickens (2000) Using the yeast three-hybrid system to detect and analyze RNA-protein interactions. Meth. Enzymol. 328: 297321.

Phizicky, E.M. & S. Fields (1995) Protein-protein interactions: methods for detection and analysis. Microbiol. Rev. 59: 94-123.

Genetic Techniques for Biological Research Corinne A. Michels Copyright © 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

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