Introduction to yeast two-hybrid

Understanding the regulation of transcription initiation by eukaryotes is the foundation of the yeast two-hybrid system. Studies have found that the initiation of cell gene transcription requires the participation of trans-transcription activators. Most transcription activators are composed of two or more independent domains, a DNA binding domain (DB) and an activation domain (AD). Later, it was discovered that the two domains not only have their own functions after separation, but also that two domains from different sources can be rebuilt and play a role in transcription activation. Fields and Song and others established the yeast two-hybrid system based on this principle.

They modeled the two proteins related to the SUC2 gene, Snf1 and Snf2, and fused the former to the Gal4 DB domain and the other to the acidic region of the Gal4 AD domain. If there is an interaction between Snf1 and Snf2, then DB and AD located on these two fusion proteins can reform the active transcription activator, thereby activating the transcription and expression of the corresponding gene. The activated gene that can show the interaction of the target protein is the reporter gene. By detecting the expression product of the reporter gene, in turn, it can be determined whether there is an interaction between the two target proteins. Here Fields et al. Used LacZ encoding β-galactosidase as a reporter gene, and introduced the GAL1 sequence regulated by Gal4 protein in the upstream regulatory region of the gene. This modified LacZ gene was integrated into the yeast chromosome URA3. Yeast's GAL4 gene and GAL80 gene (Gal80 is a negative regulator of Gal4) are deleted, thus eliminating the influence of endogenous regulators in the cell. It is known that there is an interaction between Snf1 and Snf2. It was found that only yeast cells transformed with both Snf1 and Snf2 fusion expression vectors had β-galactosidase activity, and any of these vectors alone could not detect β-galactosidase activity.

The yeast two-hybrid system mainly has two types of carriers, one is a carrier containing DNA-binding domain, and the other is a carrier containing DNA-activating domain. When constructing the fusion gene of the above two types of vectors, the test protein gene and the domain gene must be fused in reading frame. The fusion gene is expressed in the reporter strain, and its expression product can only drive the transcription of the reporter gene if it is located in the nucleus.

Most of the two-hybrid systems currently developed are based on the system established by Fields et al. These new systems have made some improvements to the reporter gene and two expression vectors. One of the important improvements is the introduction of additional reporter genes, such as the widely used HIS3 gene. After transforming yeast cells with the HIS3 reporter gene, only when HIS3 is started to express can it grow on selective media lacking histidine. Most two-hybrid systems often use two or three reporter genes simultaneously, one of which is LacZ. These modified genes have the same transcription activator binding site in the promoter region, so they can be activated by the same transcription activator (such as the Gal4 protein described above). This double or multiple selection not only improves the detection sensitivity but also reduces the false positive phenomenon.