Overview of two hybrid system for identifying and characterizing protein–protein interactions. Shown are the basic components and operation of the two hybrid system, originally devised by Fields and Song (Nature 340:245–246 [1989]) to function in the bakers yeast system. (1) A reporter gene, either a selectable marker (ie, a gene conferring prototrophic growth on selective media, or producing an enzyme for which a colony colorimetric assay exists, such as β-galactosidase) that is expressed only when a transcription factor binds upstream to a cis-linked enhancer (dark red bar). (2) A “bait” fusion protein (DBD-X) produced from a chimeric gene expressing a modular DNA binding domain (DBD; often derived from the yeast Gal 4 protein or the bacterial Lex A protein, both high-affinity, high-specificity DNA binding proteins) fused in-frame to a protein of interest, here X. In two hybrid experiments, one is testing whether any protein can interact with protein X. Prey protein X may be fused in its entirety or often alternatively just a portion of protein X is expressed in-frame with the DBD. (3) A “prey” protein (Y-AD), which represents a fusion of a specific protein fused in-frame to a transcriptional activation domain (AD; often derived from either the Herpes simplex virus VP16 protein or the yeast Gal 4 protein). This system serves as a useful test of protein–protein interactions between proteins X and Y because in the absence of a functional transactivator binding to the indicated enhancer, no transcription of the reporter gene occurs (ie, see Figure 38–16). Thus, one observes transcription only if protein X–protein Y interaction occurs, thereby bringing a functional AD to the cis-linked transcription unit, in this case activating transcription of the reporter gene. In this scenario, protein DBD-X alone fails to activate reporter transcription because the X-domain fused to the DBD does not contain an AD. Similarly, protein Y-AD alone fails to activate reporter gene transcription because it lacks a DBD to target the Y-AD protein to the enhancer. Only when both proteins are expressed in a single cell and bind the enhancer and, via DBD-X–Y-AD protein–protein interactions, regenerate a functional transactivator binary “protein,” does reporter gene transcription result in activation and mRNA synthesis (line from AD to reporter gene). Source: Chapter 39. Molecular Genetics, Recombinant DNA, & Genomic Technology, Harper's Illustrated Biochemistry, 29e Citation: Murray RK, Bender DA, Botham KM, Kennelly PJ, Rodwell VW, Weil P. Harper's Illustrated Biochemistry, 29e; 2012 Available at: https://accessmedicine.mhmedical.com/DownloadImage.aspx?image=/data/Books/murr29/murr29_c039f012.png&sec=40146142&BookID=389&ChapterSecID=40142517&imagename= Accessed: November 08, 2017 Copyright © 2017 McGraw-Hill Education. All rights reserved