Yeast Three-Hybrid System (see Zhang et al. 1999. Yeast Three-Hybrid System to Detect and Analyze Interactions Between RNA and Protein. Methods Enzymol. 306:93-113.)

A specific system to detect and analyze interactions between RNA and protein. It is similar approach to two-hybrid screen that dependes on specific protein-protein interactions (see above). The yeast three-hybrid system identifies RNA-protein interactions that are key in a large array of basic cellular processes. Examples of such processes include: translation, mRNA processing, chromosome replication and crucial decisions during early development. Moreover, all RNA viruses regulate their infectivity and replication via protein-RNA interactions. Clearly then, the yeast three-hybrid system is important in understanding the disease process related to RNA viruses, from both a basic science and clinical perspective. In practice, the yeast three-hybrid system can be used to screen RNA libraries as well as cDNA libraries, though few have been reported to date, since the technique is new.

In terms of method, a hybrid RNA binds to each of two hybrid proteins. Once this complex is formed, this activates the transcription of reporter gene. Once the reporter gene is turned on, its expression can be identified by phenotype or by simple biochemical assay. This technique can be employed if RNA and protein are each known, or can also be used to identify a "partner" when only one component is known (i.e., only the RNA or only the protein is known).

The constituents of the three-hybrid system typically include:

• A DNA-binding site that is placed upstream of a reporter gene in the yeast chromosome. [usually a 17 nucleotide recognition site for E. coli LexA protein]
• The first hybrid protein is composed of a DNA-binding domain that is fused to an RNA-binding domain. The RNA-binding domain interacts with its RNA-binding site in a hybrid RNA molecule. (Aside: hybrid denotes a "bifunctional" molecule). [hybrid protein 1 is usually LexA fused to bacteriophage MS2 coat protein]
• Next, the other part of the RNA molecule interacts with a second hybrid protein that consists of another RNA-binding domain. [hybrid protein 2 consists of the transcription activation domain of the yeast Gal4 transcription factor that is linked to an RNA-binding protein]
• However, this interaction is linked to a transcription activation domain. In essence, the hybrid RNA is "sandwiched" between two hybrid proteins. [the hybrid RNA consists of two MS2 coat-protein binding sites linked to the RNA sequence of interest, X].
• Only when all three components form a complex will the reporter gene be turned on. The product produced indicates an RNA-protein occurred, even transiently.