Two-hybrid-system

The Two-Hybrid System to Identify Proteins Involved in Specific Protein-Protein Interactions... 

Chien, C.-X, et al., 1991. The two-hybrid system A method to identify and clone genes for proteins that interact with a protein of interest. Proceedings of the National Academy of Sciences U.S.A. 88 9578 — 9582. 

Figure 5.1. Yeast two-hybrid system. Interaction of proteins X and Y upstream of a reporter gene leads to transcriptional activation. Protein X is part of a fusion protein that binds to a site on DNA upstream of the reporter gene by means of a DNA binding domain. Protein Y is part of a fusion protein that contains a transcriptional activation domain. Interaction of proteins X and Y places the activation domain in the vicinity of the reporter gene and stimulates its transcription.

A major challenge in the use of two-hybrid systems is the elimination of false positives. These clones result from activation of reporter... 

Bacterial two-hybrid system for the detection of protein-protein interactions... 

Two-hybrid system based on activation ofE. coli RNA polymerase... 

A bacterial two-hybrid system has been developed that, similar to the yeast system, functions via activation of transcription (Dove and Hochschild, 1998 Joung et al., 2000). RNA polymerase (RNAP) in E. coli consists of an enzymatic core composed of the a, (3, and (3 subunits in the stoichiometry a2(3(3, and one of several c factors that enable the enzyme to recognize specific promoters (Heilman and Chamberlin, 1988). Many bacterial transcriptional activator proteins bind the promoters they regulate and interact directly with subunits of RNAP. The most commonly observed contact is between activator proteins and the a subunit of RNAP (Ebright and Busby, 1995). The function of the a subunit is to initiate the assembly of RNAP by forming a dimer (Igarashi et al., 1991). 

Figure 5.5. A. Schematic illustration of the E. coli RNA polymerase showing the domain structure of the a subunit. The cx-NTD domain is responsible for assembly of RNAP while the a-CTD domain binds DNA and is a target for transcriptional activators. B. The two-hybrid system is based on the interaction of proteins that are fused to the X repressor and NTD domain of the a subunit of RNAP. In the example shown, Gal4 interacts with Gall IP to recruit RNAP to the promoter and activate transcription of the lacZ reporter gene. Figure adapted from Dove and Hochschild (1998).

The bacterial one and two-hybrid systems have potential advantages over the yeast two-hybrid system due to the higher transformation efficiency and faster growth rate of coli. To date, however, the bacterial two-hybrid system has not been used for genome-scale analysis of protein-protein interactions. 

The experiments described above indicate that technology is available to couple SPR with mass spectrometry. These methods should be useful for protein-protein interaction mapping. For example, immobilized proteins can be used as hooks for fishing binding partners from complex protein mixtures under native conditions. The coupling of techniques can lead not only to the rapid identification of interacting proteins but will also provide information on the kinetic parameters of the interaction. This approach should serve as an excellent complement to the use of in vivo techniques such as the yeast two-hybrid system. 

Bai, C., and Elledge, S. J. (1997). Gene identification using the yeast two-hybrid system. Methods Enzymol. 283, 141-156. 

Legrain P et al. Genome-wide protein interaction maps using two-hybrid systems. FEBS Lett 2000 480 32-36. 

Pazos, F. and Valencia, A. (2002) In silico two-hybrid system for the selection of physically interacting protein pairs. Proteins 47, 219-227. 

M. C., Yu, C. H., Lin, P. I., and Wu, W. F. Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system. /. Bacteriol. 2003, 185, 2393-2401. 

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