vations, Facts sheet, Small Molecule Therapeutics, Inc.). In contrast to ToxR, CadC is a naturally occurring single transmembrane protein in E. coli that signals in response to pH change. These simple E. coli systems are modular in that chime-ric proteins can easily be developed with the CadC protein. In addition, these systems can be easily adapted to identify compounds that induce or prevent dimerization.
Proteins carry out functions by interacting with other proteins. The ToxR and CadC protein dimerization systems can be used to measure homodimeric interactions of membrane-associated proteins. There are other E. coli-based systems that can be used for measuring heterodimeric interactions. A system developed by Dove et al. [55,56] uses transcriptional activators in prokaryotes that bind near a promoter and contact RNA polymerase. RNA polymerase consists of the p, P1, o, and two a subunits. Each a subunit contacts an activator protein, such as ^ci, two of which are required to occupy the ^-operator sites and activate the promoter. The C-terminal end of the RNA polymerase a subunit is fused to one protein of the interacting pair, and the C-terminal end of the ^cl protein is fused to the second protein of the interacting pair. When contact is made between the protein fused to the DNA-bound protein, Xcl, and the heterologous protein domain fused to RNA polymerase, transcription is activated. There are many examples of the activation of genes by recruitment of proteins , and many of these can be applied to developing protein-protein interaction screens. Another novel E. coli two-hybrid system has been developed that takes advantage of the ability of LexA to repress the activator AraC . Chimeras of AraC and LexA were made with the interacting proteins. The LexA-protein 1 chimera interacts with the LexA binding half-site on the DNA, and the AraC-protein 2 chimera binds to the high-affinity AraC binding site. The LexA and AraC operators are separated by an IHF site that is involved in DNA loop formation and possibly helps in repression. Interaction of protein 1 and protein 2 allows LexA to hetero-dimerize with AraC, causing repression of the AraBAD promoter fused to lacZ.
Another E. coli two-hybrid system that can be used for homo- and hetero-dimeric proteins has been described . This particular system is simple in that reconstitution of enzyme activity is used. The genes of the two interacting proteins of interest are fused to the two fragments of the catalytic domain of Borde-tella pertussis adenylate cyclase. One fragment with amino acid 1-224 of the cyclase is constructed with a C-terminal fusion and the second with amino acid 225-399 is constructed with an N-terminal protein fusion. Interaction of the proteins reconstitutes the adenylate cyclase and results in cAMP synthesis in E. coli with a mutation in the cya gene. cAMP binds to the catabolite gene activator protein, CAP, and the cAMP/CAP complex can bind specific promoters to turn on certain genes. Reporters of interest, such as lacZ or chloramphenicol resistance genes, can be fused to the cAMP sensitive reporters to obtain an easily measurable readout. Protein-protein interaction can also be measured by fusing the proteins of interest to complementing P-galactosidase deletion mutants . The forced interaction of nonfunctional P-galactosidase units to produce active enzyme is the basis of this technology and provides a second method to measure proteinprotein interaction directly without the activation of transcription factors.
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