Yeast three-hybrid

Yeast three-hybrid

Many biological processes are realized through the interaction of macromolecules such as protein-protein and nucleic acid-protein. The study of the interaction between these macromolecules helps to reveal the molecular mechanism of life process. The yeast two-hybrid system is a useful tool for studying the interaction between proteins. With the wide application of the system, a three-hybrid system has been developed on this basis to study the interaction of more complex macromolecules including three components. The study of protein-protein, RNA-protein and small molecule-protein interactions provides new tools.

The yeast three-binding hybrid system is mainly used to analyze the interaction between protein and RNA. Senguptaxi et al. First reported the use of a three-hybrid system to analyze the interaction between metal regulatory proteins and metal response element RNA sequences, as well as HIV transcription activation proteins and HIV transcription activation response element RNA sequences.

The basic principle of the yeast three-hybrid system is to fuse a known RNA binding protein with the DNA binding domain of a transcription factor to construct the first fusion protein, and the second protein (the RNA binding protein to be selected) is fused to the transcription activation domain Construction of fusion proteins. In addition, a hybrid RNA was constructed and expressed, containing two different binding sites. When RNA interacts with the binding sites of two RNA-binding proteins, it can activate the transcription and expression of the reporter gene. For example, in the study of the interaction between IRP1 and IRE RNA, Dhruba et al. Used the bacteriophage MS2 coat protein as the first RNA-binding protein. MS2 coat protein can recognize the 21-base stem-loop sequence in RNA And have a higher affinity with it. It was fused with LexA to construct a hybrid protein. The activation domain of IRP1Gal4 is fused to construct another hybrid protein. IRE mRNA has a 21-base stem-loop sequence in the non-coding region, and the coding region encodes a protein sequence that specifically binds to IRP1. In the experiment, they used a plasmid to express hybrid RNA, containing 2 MS2 capsid protein binding sites and 1 IRE.

The yeast three-hybrid system provides a fast and versatile new method for detecting RNA-protein interactions in vivo. Compared with the two-hybrid system, hybrid RNA and hybrid protein are somewhat different: First, the hybrid RNA molecule may not be a physiological structure, the different RNA components of the hybridization will have a certain effect on the normal structure. However, Dhruba's research shows that local and relatively stable structural regions such as MS2 capsid protein and IRP1 recognition sites can coexist in the same hybrid RNA molecule in vivo. Secondly, the structure of the stem-loop region in these RNAs is quite stable, as long as the RNA forms part of the correct structure, it is sufficient to mediate transcriptional activation.

The yeast three-hybrid system can be used to analyze and determine the precise domain of RNA-protein interactions, even single nucleotide or amino acid residues; it can also be used to identify and clone RNA with important physiological functions (such as transcription, positioning, RNA virus Packaging and infection, etc.) protein, can be used in the mechanism of regulation, disease prevention and development of antiviral drugs; you can also use this system to screen RNA that binds to specific proteins. It is also possible to develop a four-hybrid system on this basis to study RNA-RNA interactions.