What is yeast two-hybrid system?

Posted by beauty33 on April 25th, 2019

In the yeast two-hybrid system, the two proteins to be studied are separately cloned (fused) into the DNA binding domain (DNA-BD) and the transcription activation domain (AD) of a transcriptional activator (such as GAL4) of the yeast expression plasmid, and constructed. A fusion expression vector is used to analyze the system of two protein interactions from the expression product.

Introduction

The establishment of the yeast two-hybrid system is based on the understanding of the transcriptional initiation process regulated by eukaryotes. Transcription of a cell initiation gene requires the involvement of a trans-transcriptional activator. A trans-transcriptional activator, such as the yeast transcription factor GAL4, is structurally modular and is often composed of two or more structurally separate, functionally independent domains, among which DNA A binding domain (DNA-BD) and an activation domain (DNA-AD). These two binding domains still function separately when they are separated, but they cannot activate transcription. Only when the separated two are spatially close by appropriate pathways can the complete GAL4 transcription factor be re-presented and activated. A downstream promoter of the upstream activating sequence (UAS) that transcribes genes downstream of the promoter.

The establishment of a two-hybrid system is based on the understanding of the transcriptional initiation process regulated by eukaryotes. Transcription of a cell initiation gene requires the involvement of a trans-transcriptional activator. Work in the 1980s showed that transcriptional activators are structurally modular, that is, these factors are often composed of two or more independent domains, including a DNA binding domain (DNA binding domain). BD) and activation domain (AD), which are required for transcriptional activators to function. Although BD alone binds to the promoter, it does not activate transcription. Hybrid proteins formed by BD and AD of different transcriptional activators still have a normal function of activating transcription. Hybrid proteins, such as the fusion of BD of the Gal4 protein of yeast cells with an acidic activation domain B42 of E. coli, can still bind to the Gal4 binding site and activate transcription.

In the yeast two-hybrid system, the "bait" protein X is cloned into a DNA-BD vector to express a DNA-BD/X fusion protein; the protein Y to be tested is cloned into an AD vector to express an AD/Y fusion protein. Once there is an interaction between the X and Y proteins, DNA-BD and AD are also pulled closer, returning to function, and activating the expression of the LacZ and HIS3 genes in the recombinant.

Technical Procedure

1. The bait plasmid is constructed by fusing a gene to be tested with a DNA binding domain of Gal4 or LexA or other suitable protein;
2. transforming the bait plasmid into a yeast cell strain lacking the reporter gene promoter, and selecting the transformed yeast;
3. Convert the library plasmid into yeast;
4. Screen for interacting proteins by reporter gene function.

Research process

The work of Fields et al. marks the official establishment of the two-hybrid system. They modeled the two proteins Snf1 and Snf2, which are involved in the regulation of the SUC2 gene, and fused the former with the BD domain of Gal4. The other fusion with the acidic region of the AD domain of Gal4 is generally referred to as the fusion protein formed by BD and AD. It is a "bait" and "prey or target protein". If there is an interaction between Snf1 and Snf2, BD and AD, respectively located on the two fusion proteins, can re-form the active transcriptional activator, thereby activating the transcription and expression of the corresponding gene. This activated gene, which shows the interaction between "bait" and "prey", is called a reporter gene. By detecting the reporter gene expression product, it is in turn possible to discriminate whether there is an interaction between the two proteins as "bait" and "prey". Here, Fields et al. used LacZ encoding β-galactosidase as a reporter gene and introduced a Gal4 protein-regulated GAL1 sequence in the upstream regulatory region of the gene. This engineered LacZ gene was integrated into the URA3 position of the yeast chromosome. The yeast GAL4 gene and the GAL80 gene (Gal80 is a negative regulator of Gal4) were deleted, thereby eliminating the influence of cellular endogenous regulators. It is known that there is an interaction between Snf1 and Snf2. As a result, it was found that only yeast cells which simultaneously transformed the Snf1 and Snf2 fusion expression vectors had β-galactosidase activity, and any of the vectors alone could not detect β-galactosidase activity.

Application

The yeast two-hybrid system is capable of determining the binding of proteins in vivo and is highly sensitive.

It is mainly due to:

1 The expression vector of high copy and strong promoter is used to overexpress the hybrid protein.

2 Signal measurement is carried out under natural equilibrium concentration conditions, and physical methods such as immunoprecipitation require multiple washings to achieve this condition, reducing signal intensity.

3 Hybrid interprotein stability can be enhanced by the binding of the activation domain and the binding domain to form a transcription initiation complex, which in turn binds to the promoter DNA, which conjugates to stabilize the binding of each component.

4 Amplification of the signal by the production of a variety of stable enzymes by the mRNA. At the same time, the yeast phenotype, X-Gal and HIS3 protein expression and other detection methods are very sensitive.

Reverse two-hybrid system

In the study of the structural features and modes of action of proteins, it is sometimes necessary to destroy the interaction between proteins by means of mutations and addition of inhibitors. In response to this need in practice, Vidal et al. developed the so-called reverse two-hybrid system. The key to this technology is the introduction of the reporter gene URA3. The URA3 gene plays a counter-selection role here, and the enzyme encoded by it is a key enzyme for uracil synthesis. The enzyme converts 5-fluoroorotic acid (5-FOA) into a substance that is toxic to cells. Vidal et al. introduced a binding site for Gal4 within the promoter of the URA3 gene by engineering. This engineered yeast strain can only grow in the selective medium lacking uracil only when the "bait" and "prey" interact to activate the expression of the URA3 gene. The interaction of "bait" and "prey" on complete medium containing 5-FOA inhibits cell growth. However, if the protein of interest, i.e., the protein fused to DB or AD, is mutated or does not interact due to interference with the drug, and the URA3 gene is not expressed, the cells can be grown on complete medium containing 5-FOA. In this way, Vidal et al. screened for mutations in the transcription factor E2F1, which still bind to the retinoblastoma protein RB but lose the ability to bind to another protein called DP1. The results were verified by in vitro binding experiments. By sequencing these mutant protein genes, they discovered a new site where E2F1 binds to DP1.