Homework SourseThe yeast twohybrid system depends on the modular nature of
The yeast two-hybrid system depends on the modular nature of many transcription factors, which have one domain that binds to DNA and another domain that activates transcription. Domains can be interchanged by recombinant DNA methods, allowing hybrid transcription factors to be constructed. Thus, the DNA-binding domain of the E. coli LexA repressor can be combined with the powerful VP16 activation domain from herpesvirus to activate transcription of genes downstream of a LexA DNA-binding site.
If the two domains of the transcription factor can be brought into proximity by protein-protein interactions, they will activate transcription. This is the key feature of the two-hybrid system. Thus, if one member of an interacting pair of proteins is fused to the DNA-binding domain of LexA (to form the ‘bait’) and the other is fused to the VP16 activation domain (to form the ‘prey’), transcription will be activated when the two hybrid proteins interact inside a yeast cell. It is possible to design powerful screens for protein-protein interactions, if the gene whose transcription is turned on is essential for growth or can give rise to a colored product.
To check out the ability of the system to find proteins with which Ras interacts, hybrid genes were constructed that contained the LexA DNA-binding domain, one fused to Ras (LexA-Ras) and the other fused to nuclear lamin (LexA-lamin). A second pair of constructs contained the VP16 activation domain alone (VP16) or fused to the adenylyl cyclase gene (VP16-CYR). Adenylyl cyclase is known to interact with Ras and serves as a positive control; nuclear lamins do not interact with adenylyl cyclase and serve as a negative control. These plasmid constructs were introduced into a strain of yeast containing copies of the His3 gene and the LacZ gene, both with LexA-binding sites positioned immediately upstream. Individual transformed colonies were tested for the ability to grow on a plate lacking histidine, which requires expression of the His3 gene. In addition, they were tested for ability to form blue colonies (as compared to the normal white colonies) when grown in the presence of an appropriate substrate (XGAL) for -galactosidase. The setup for the experiment is outlined in Table 1.
A.) Fill in the table with your expectations. Use a + sign to indicate growth on plates lacking histidine and a – sign to indicate no growth. Write ‘blue’ or ‘white’ to indicate the color of colonies grown in the presence of XGAL.
B.) For any entries in the table that you expect to grow in the absence of histidine and form blue colonies with XGAL, sketch the structure of the active transcription factor on the LacZ gene.
C.) If you want two proteins to be expressed in a single polypeptide chain, what must you be careful to do when you fuse two genes together?
Table 1:
Plasmid constructs
Growth on plates lacking histidine
Growth on plates with XGAL
Bait
Prey
LexA-Ras
LexA-lamin
VP16
VP16-CYR
LexA-Ras
VP16
LexA-Ras
VP16-CYR
LexA-lamin
VP16
LexA-lamin
VP16-CYR
| Plasmid constructs | Growth on plates lacking histidine | Growth on plates with XGAL | |
| Bait | Prey | ||
| LexA-Ras | |||
| LexA-lamin | |||
| VP16 | |||
| VP16-CYR | |||
| LexA-Ras | VP16 | ||
| LexA-Ras | VP16-CYR | ||
| LexA-lamin | VP16 | ||
| LexA-lamin | VP16-CYR |
Solution
Transcription factor has two domains. One is for DNA binding and another for VP16 activation.
One protein is fused to the DNA-binding domain of LexA repressor to form bait.
Another protein is fused to the VP16 activation domain of LexA repressor to form prey.
If these two proteins interact with each other inside the yeast cell, the activation of Lac Z gene transcription will take place.
VP-16-adenylyl cyclase interacts with LexA binding domain-Ras to act as positive control
VP-16-adenylyl cyclase interacts with LexA binding domain-Lamin to act as negative control
The plasmid constructs with these modifications can be put into yeast cells consisting of His3 gene and lacZ gene
Growth Plate that lacks histidine allows the growth of cells with His3 gene. The plate that have blue colonies indicate that the cell consists of beta-galactosidase gene that can convert XGAL as substrate.
Table 1:
Plasmid constructs
Growth on plates lacking histidine
Growth on plates with XGAL
Bait
Prey
LexA-Ras
-
white
LexA-lamin
-
white
VP16
-
white
VP16-CYR
-
white
LexA-Ras
VP16
+
blue
LexA-Ras
VP16-CYR
+
blue
LexA-lamin
VP16
+
blue
LexA-lamin
VP16-CYR
-
white
| Plasmid constructs | Growth on plates lacking histidine | Growth on plates with XGAL | |
| Bait | Prey | ||
| LexA-Ras | - | white | |
| LexA-lamin | - | white | |
| VP16 | - | white | |
| VP16-CYR | - | white | |
| LexA-Ras | VP16 | + | blue |
| LexA-Ras | VP16-CYR | + | blue |
| LexA-lamin | VP16 | + | blue |
| LexA-lamin | VP16-CYR | - | white |
