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Yeast n-Hybrid Systems for Molecular Evolution

As outlined in Fig. 7.1, the Y2H system consists of two protein chimeras, and a reporter gene downstream from the binding site for the transcriptional activator. If the two proteins of interest (X and Y) interact, they effectively dimerize the DNA-binding protein chimera (DBD-X) and the transcription activation protein chimera (AD-Y). Dimerization of the DBD and the transcription AD helps to recruit the transcription machinery to a promoter adjacent to the binding site for the transcriptional activator, thereby activating transcription of the reporter gene. [Pg.127]

The assay was demonstrated initially using two yeast proteins known to be physically associated in vivo [1]. The yeast SNF1 protein, a serine-threonine protein kinase, was fused to the Gal4 DBD, and the SNF1 activator protein SNF4 was fused to the Gal4 AD. [Pg.127]

Compared with the Y2H system, the Y3H system used to detect RNA-protein interactions has one additional component, a hybrid RNA molecule [90], One half of the hybrid RNA is a known RNA (R) that can binds the MS2 coat protein (MS2) with high affinity and serves as an anchor. The other half is RNAX, whose interaction with protein Y is being tested. D. Another version of the Y3H system can be used to detect small molecule-protein interactions [96], Ligand LI which interacts with protein X can be linked to ligand L2, and thus, if L2 interacts with Y, transcriptional activation of the reporter gene will be reconstituted. [Pg.128]

Since the initial paper by Fields and Song, there have been significant technical improvements in the method. DNA-binding domains and transcription activation domains have been optimized to reduce false positives and increase the transcription read-out. A variety of reporter plasmids have been engineered to detect a broad range of protein-protein interactions. Much more is understood about the nature of false positives and how to rout them out. Moreover, in response to the utility of this approach, several laboratories have begun to develop transcription-based assays that can be carried out in bacteria, or protein-protein interaction assays based on alternate readouts such as enzyme complementation or fluorescence resonance energy transfer (FRET). [Pg.129]

While envisioned initially as a method for verifying positive protein-protein interactions, it was quickly realized that the method was well suited to screening libraries of proteins to identify new interactions [5], Now in widespread use, the Y2H assay has [Pg.129]


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