The yeast one-hybrid (Y1H) system enables the identification of proteins that can bind to DNA elements of interest, including cis-regulatory elements, origins of DNA replication, and telomeres. In the Y1H system, using a single hybrid protein AD-Y, reporter gene expression is activated when Y interacts with the DNA bait. Although many of the predicted regulatory TF contain intrinsic AD, some TF have a repressor domain or no activation / repressor domain at all. In addition, DNA-binding proteins that do not function in transcription (e. g., replication and DNA repair proteins) do not contain AD. To be able to identify various DNA binding proteins, strong heterologous AD was added to the prey proteins. To facilitate the high-throughput, unbiased identification of protein – DNA interactions, we have developed a Y1H system compatible with Gateway. This system can be used for small copies (e. g. cis-regulatory elements) and single copies of large DNA fragments (e. g. gene promoters). In many cases, DNA binding proteins are often regulated by the recruitment of accessory proteins. Most of bZIP and bHLH, families, such as Jun-Fos, Myc-Max as well as classical phage λ repressor and Cro proteins, belong to this class of transcription factors. Many yeast genetic methods have been reported to study proteins in complexes with known chaperones to form heterodimeric complexes that bind DNA; these complexes may include a chaperone that lacks intrinsic DNA binding capacity through dimerization with accessory proteins. Traditionally, these studies have used two separate plasmids to express two different proteins. Researchers have developed a plasmid-based modified Y1H system (MY 1 H). In addition to the AD fusion protein, the second protein was also co-expressed at comparable or excess levels.

The yeast one-hybrid system (Y1H) provides one of the few direct strategies, often used to identify transcription factor (TF) -promoter interactions, with a focus on individual promoter regions. This method requires two main components: a reporter construct (bait) that carries a promoter region (e. g. lacZ), usually integrated into the genome of the yeast strain (such as YM4271) and an effector construct (prey) driving the expression of a TF (Gal 4-AD) (AD). After converting the effector constructs into reporter strains, increased reporter activity revealed TF-promoter interactions depending on the DNA binding affinity and specificity of the TF-AD effectors for the promoter baits. Following the initial design, the effector constructs used for Y1H screening were part of the cDNA library. These cDNA libraries provide only partial and biased libraries of potential DNA binding proteins. With the era, some efforts have been made to develop global TF effector libraries for different species. Adjusting the Y1H system to these TF ORFeome clone collections significantly improved the method, enhancing the discovery of functional TF-promoter interactions in an unbiased and comprehensive manner.Overall, the yeast one-hybrid system constructs an expression plasmid for a fusion of a library protein to a transcription activation domain as well as a reporter plasmid, and utilizes biological activity to detect DNA – protein interactions.

Yeast single-hybridization rapidly screens numerous transcription factors and significantly accelerates the research process. With the help of the high-throughput library construction strategy, this technology can cover a wide range of transcription factors, providing a strong support for exploring the regulatory network of specific genes. The experiments were conducted in yeast cells near the natural state, ensuring that the obtained interaction data are highly physiological relevant, thus more accurately reflecting the interaction between protein and DNA in a real cell environment. This technique can not only verify the existence of protein-DNA interactions, but also quantify the strength of interactions by assessing the expression level of reporter genes, opening a way for deep insights into the quantitative features of protein-DNA interactions. The yeast single-hybrid system is easy to operate and standardized, which is easy to be used in various laboratory environments. In addition, yeast cell culture and screening costs are low, so the technology shows good economy. In the regulation of gene expression in eukaryotic cells, yeast one-hybrid technology can be used to identify DNA binding sites, excavate potential binding protein genes, and resolve DNA binding domain information. By monitoring the phenotypic changes in reporter genes, this technique can dissect the interaction between DNA and proteins, and then reveal the mechanism of gene regulation involved in eukaryotic cells. Given the high sensitivity and reliability of the yeast one-hybrid method in detecting specific interactions between specific transcription factors with cis-acting elements, it has become the method of choice for cloning specific transcription factors of low abundance and difficult to purify by biochemical means. The yeast single-hybrid technique can be used to determine whether there are known interactions between DNA and protein. Yeast single-hybrid technology also has potential applications in new drug development. By constructing a yeast one-hybrid system for drug targets, the screening of drug molecules that interact with the target can be screened, providing strong support for new drug development.

The yeast one-hybrid system (Y1H), as a derivative of the yeast two-hybrid system (Y2H), is a versatile and widely used in vivo genetic analysis tool designed to identify interactions between proteins and DNA. Y1H enables not only to isolate protein-coding genes bound to cis-acting elements, but also enables in-depth characterization of known protein – DNA interactions, while the Y2H system focuses on the detection of protein-protein interactions. In the Y2H system, the construction of two hybrid proteins is involved: the bait protein (X) is fused to the DNA binding domain (DB), and the prey protein (Y) is coupled to the transcriptional activation domain (AD). When X and Y physically interact with each other, the functional TF is reconstructed and reporter gene expression is activated. In contrast to Y2H, Y1H directly obtains the nucleotide sequence encoding a protein bound to DNA directly from a gene library without complex protein isolation and purification operations. Provides efficient methods for cloning DNA binding proteins that facilitate the identification of proteins that interact with specific DNA sequences. Has unique advantages in studying the expression regulation of genes within eukaryotic cells.In conclusion, the yeast one-hybrid system and the yeast two-hybrid system each have their own unique principles and advantages, and which system selection depends on the specific research purpose and experimental conditions.

In yeast one-hybrid experiments, we encounter bait protein functions that may be impaired by truncation. Insufficient chemical conversion or electroconversion efficiency, and too few or too many positive clones. Facing the above problems, TEK Biotech ensured that the number of clones screened was greater than 1 million. TEK Biotech adjusted AbA inhibitory concentration, such as increased from 100 ng / ml to 200 ng/ml rescreen.When performing the interception of transcription activation region, the operation should be careful to prevent the interaction results. At the same time, the application of 3 ′ AT or ABA can be considered to inhibit the background signals, but we should be alert to the possible toxic effects of these two chemical reagents on yeast growth. In terms of chemical transformation, TEK Biotech ensures that the transformation efficiency of competent cells is not less than 10 ^ 4, and one way to improve the transformation efficiency is to increase the amount of oxygen of competent cells, the specific operation is to control the amount of liquid not more than one fifth of the volume of the container. As for the electrical conversion, it adds 10% glycerol to preserve the competent cells, and sets the conversion parameters to 800 volts and 15 ms, so that the conversion efficiency can be up to 10 ^ 5 to 10 ^ 6 times.