Figure 20-29. Yeast two-hybrid system for detecting proteins that interact.

Figure 20-29Yeast two-hybrid system for detecting proteins that interact

(a) Recombinant DNA techniques can be used to prepare genes that encode hybrid (chimeric) proteins consisting of the DNA-binding domain (purple) or activation domain (orange) of a transcription factor fused to one of two interacting proteins, referred to as the “bait” domain (pink) and “fish” domain (green). (b) If yeast cells are transfected with genes encoding both hybrids, the bait and fish portions of the chimeric proteins interact to produce a functional transcriptional activator. One end of this protein complex binds to the upstream activating sequence (UAS) of a test gene (in this example, the HIS3 gene); the other end, consisting of the activation domain, stimulates assembly of the transcription-initiation complex (gray) at the promoter (yellow). (c) This strategy can be used to screen a cDNA library for clones expressing proteins that interact with a protein of interest, in this case Ras. This approach requires two types of plasmids: The bait plasmid includes a DNA sequence encoding the DNA-binding domain of a transcription factor (purple) connected to the coding sequence for Ras (pink). The fish plasmids contain individual cDNAs (green) from a library connected to the coding sequence for the activation domain (orange). Each type of plasmid also contains a wild-type selection gene (e.g., TRP1 or LEU2). Both types of plasmids are transfected into yeast cells with mutations in genes required for tryptophan, leucine, and histidine biosynthesis (trp1, leu2, his3 cells) and then grown in the absence of tryptophan and leucine. Only cells that contain the bait plasmid and at least one fish plasmid survive under these selection conditions. The cells that survive then are plated on medium lacking histidine; only cells that contain the bait plasmid and a fish plasmid encoding a protein that binds to Ras are able to grow, thus identifying cDNAs encoding Ras-binding proteins. [See A. B. Vojtek et al., 1993, Cell 74:205; S. Fields and O. Song, 1989, Nature 340:245.]

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From: Section 20.5, MAP Kinase Pathways

Cover of Molecular Cell Biology
Molecular Cell Biology. 4th edition.
Lodish H, Berk A, Zipursky SL, et al.
New York: W. H. Freeman; 2000.
Copyright © 2000, W. H. Freeman and Company.

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