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: