Yeast two-hybrid library screening.

Abstract

The two-hybrid system was originally devised by Fields and Song () as a protein interaction detection system in yeast. Subsequently, it has been employed in many laboratories as a means of screening cDNA and genomic fusion libraries for protein interaction partners (, , , , , , ). The method relies on the fact that transcription factors, such as the yeast GAL4 factor, consist of separable DNA-binding and transcriptional regulatory domains; the former being required to direct the latter to appropriate promoters where transcriptional activation is effected, usually by direct or indirect interaction of the activation domain with the general transcription machinery. The essence of the two-hybrid system is the in vivo reconstitution of a functional transcriptional activator from two interacting polypeptides, one fused to a sequence-specific DNA-binding domain and the other to a potent transcriptional activation domain. Interaction is detected when the reconstituted transcription factor activates a reporter gene (see Fig. 1). DNA-binding domain and activation domain fusion proteins are expressed from plasmid DNAs. The DNA-binding and transcriptional activation components are usually derived from the yeast GAL4 transcription factor (see Figs. 1 and 2), although alternatives have been used by some workers (,,). Where the GAL4 DNA-binding domain is used, yeast strains in which the wild-type GAL4 gene has been deleted are employed. For the purposes of identifying new protein partners, DNA encoding the polypeptide for which partners are sought (bait polypeptide) is ligated into a yeast shuttle vector to create a DNA-binding domain-bait fusion (Fig. 2A). Library cDNA is ligated into a second shuttle vector to create an activation domain-tagged cDNA library (see Fig. 2B). In the original method as proposed by Chien et. al. (), the bait (GAL4 DNA-binding domain) construct and activation domain fusion library were cotransformed into a yeast strain containing an integrated LacZ gene driven by a GAL4-responsive promoter (). Yeast-containing plasmids encoding interacting polypeptides were scored by the presence of β-galactosidase activity (blue colonies in the presence of the chromogenic substrate X-Gal). The method was subsequently developed to facilitate the screening of large mammalian cDNA libraries (). This innovation employs a his3 test yeast strain (that is auxotrophic for histidine) that contains, in addition to the lacZ reporter, an integrated HIS3 gene that is transcribed at a significant level only in the presence of the reconstituted activator (see Fig. 2). Yeasts transformed with the bait plasmid and the activator library are plated on minimal plates lacking histidine. Only HIS3 colonies are then carried forward for further analysts. The genotype of two commonly used yeast reporter strains is given in Note 1. Open image in new window Fig. 1. The principle of the yeast two-hybrid system. The two-hybrid system is a method of detecting specific protein-protein interactions between two polypeptides in vivo. The “bait” and “target” polypeptides are fused to the DNA-binding and transcriptional activation domains of the transcription factor GAL4, respectively (other DNA-binding and activation domains have been used). In vivo interaction of “bait” and “target” polypeptides results in formation of a functional activator and activation of GAL4-responsive reporter genes. Yeast strains used for two-hybrid analysis, such as YPB2 and HF7c, contain two integrated GAL4-responsive reporter genes HIS3 and LacZ. Activation of the HIS3 reporter gene confers the ability to grow on media lacking histidine. (A) In the situation where bait and target proteins do not interact, the bait polypeptide is targeted to the promoter of the HIS3 reporter gene through specific protein-DNA interactions between the GAL4 DNA-binding domain (DBD) and the GAL1 UAS, but the activation domain is not recruited to the promoter, resulting in no transcription of the HIS3 gene. (B) Where there is a functional interaction between bait and target polypeptides, a functional activator is formed on the GAL1 UAS; HIS3 expression ensues, resulting in histidine prototrophy. (C) The LacZ reporter gene in strains, such as YPB2 or HF7c, is driven by an artificial GAL4-dependent promoter. Interaction of bait and target polypeptides results in expression of β-galactosidase activity, which is easily assayed. Open image in new window Fig. 2. Cloning sites in the DNA-binding domain fusion vector pGBT9 and activation domain vector pGAD424. (A) The region of pGBT9 around the segment encoding the DNA-binding domain of GAL4 (residues 1–147). P ADH1 , ADH1 promoter; MCS, multicloning site; T ADH1 , ADH1 transcriptional terminator. The TRP1 aux-atrophic marker gene is also indicated. The first TCG codon in the following sequence corresponds to residue 147 of GAL4. Restriction sites are indicated as are termination codons (Term). (B) The region of pGAD424 around the segment encoding the GAL4 activation domain (residues 768–881). The first GAG codon in the sequence corresponds to codon 881 of GAL4. The segment designated N indicates the SV40 large T nuclear localization sequence engineered into the N-terminus of GAL4 ().