Transcription factor reconstitution

The Sos recruitment yeast two-hybrid system was developed by Aronheim and colleagues (1997). It is now known as the CytoTrap yeast two-hybrid system and marketed by Stratagene (La Jolla, California, USA). The CytoTrap system differs from both the GAL4 and the LexA systems in that it is not dependent on transcription factor activation in the nucleus for the detection of protein-protein association. Instead, protein interactions are detected in the cytoplasm and involve the reconstitution of the Sos/Ras signaling pathway in conjunction with the temperature-sensitive yeast strain, cdc25H. 

Extensive studies on the reconstitution of a regulated transcription initiation in in vitro systems have shown that most of the specific transcription factors are not capable of stimulating transcription above the basal level without the assistance of further proteins. Further coactivators are required for this task. The coactivators can be subdivided into three classes (Fig. 1.34). 

LexA (3). The second hybrid, referred to as the prey, consists of a putative interacting protein fused to the activation domain of this transcription factor. Physical interaction between bait and prey will bring the transcription factor DNA-binding domain and activation domain into close proximity of each other, thereby reconstituting a transcriptionally active chimera transcriptional activity can be easily monitored through a convenient reporter system or selection scheme in yeast (e.g., lacZ) (1, 3). 

Activators can only elicit a very low level of activation in a transcription system reconstituted from pure RNA polymerase 11 (pol 11) and basal factors, some types of co-factor are needed in addition. One such factor was found and termed Mediator. In an in vitro transcription system reconstituted from essentially pure factors, the mediator complex was shown to stimulate basal transcription 50-fold and to support transcriptional activation from the Gal4-VP16 and Gcn4 activating protein. The Mediator complex could also be purified in a complex with RNA polymerase II (pol II), thus forming a holo-enzyme form of pol II. 

A transcription-competent pre-initiation complex consisting of general transcription factors and RNA polymerase II can be reconstituted in the test tube from the individual components. As outlined in Fig. 1.20B, efficient reconstitution requires a defined order for the addition of the individual components. 

The yeast two-hybrid system is a powerful genetic technique that has been used successfully to study protein-protein interactions in vivo from a wide variety of biological sources, including yeast, plant, and mammalian (Fields and Song, 1989). The two-hybrid system provides a sensitive method to detect relatively weak and transient protein-protein interactions that may not be biochemically detectable. The principle of this assay is based on the reconstitution of transcriptional factors that promotes the expression reporter genes, which then allow the proliferation of yeast under restrictive conditions. Using the two-hybrid system, we successfully isolated IPCEFl and a novel kinesin motor proteins from a rat brain cDNA library as interactors of cytohesin 2 ARF GEF and centaurin-al ARF GAP, respectively (Venkateswarlu, 2003). The procedure used to map the binding domains of cytohesin 2 and IPCEFl, and to characterize the cytohesin 2 and IPCEFl selectivity is outlined here. 

Figure 3 Schematic illustrating the reverse yeast two-hybrid method for PPI detection, (a) In the absence of any inhibitors, the reconstituted transcription factor leads to the expression of a cytotoxic product, and cell death, (b) In the presence of an inhibitor that targets the PPI, transcription is inhibited, and the cell survives.

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