Genomic Analysis Utilizing the Yeast Two-Hybrid System

Abstract

As the completion of genome sequencing efforts leads to the definition of increasing numbers of genes, the need to reliably assign function to identified coding sequences becomes paramount. One means of gaining initial insight into the function of an undefined protein is to develop a map of other defined proteins with which it physically or functionally interacts. There are several approaches to assigning interacting protein groups. In suitable model organisms such as yeast, a traditional approach has been to create null mutations in the gene encoding the novel protein of interest, and to use suppressor analysis to identify genetically (functionally) interacting proteins. Alternatively, copurification of complexes of interest followed by use of mass spectrophotometry to assign identity of individual component proteins has been used to define interacting groups based on physical interactions. The genetic approaches offer speed and low cost; the physical approaches offer the certainty that copurified proteins physically function together on the protein level, rather than being connected via indirect regulatory pathways. A third approach, the yeast two-hybrid system, combines the advantages of working with yeast while targeting proteins that physically associate.