Gene Cloning

298 Gene Cloning
1
AD
BD
Activation of
transcription
UAS GAL
promoter
Gal gene
2
AD
SNF4
SNF1
BD
Activation of
transcription
UAS GAL
promoter
Gal gene
Figure 10.9 The two-hybrid assay in yeast. 1) shows the two domains of
the Gal4 protein. BD, the binding domain, positions the Gal4 upstream of the
GAL promoter. AD, the activation domain, activates transcription of the GAL
gene. 2) Shows that the BD and AD of Gal4 can be separated. If they are fused
to two proteins which themselves interact – here, SNF1 and SNF4 – activation
of transcription of the downstream gene will still occur.
researchers to test an idea which at first sight seemed far-fetched: would
the process of gene activation still work if the two parts of the Gal4 were
actually on separate proteins, but these two proteins themselves contacted
each other?
To test this idea, the following experiment was done. A fusion was made
between a GAL promoter and a lacZ gene, thus placing lacZ under the control
of the GAL promoter. This was done in a host strain where the endogenous
gal4 gene had been removed, so the cells were unable to respond to
galactose in the medium. Two translational fusions were then cloned onto
two separate plasmids. In one, the part of the gal4 gene encoding the N-
terminal end of the Gal4 protein, which is the part of the protein that binds
to the UAS sequence, was fused to the 5′ end of a gene called SNF1, which
encodes a protein kinase. In the other, the part of the gal4 gene encoding
the activator domain of Gal4 was fused to the 3′ end of a gene encoding
SNF4, which was known from other studies to physically interact with
SNF1. The two constructs were introduced into the yeast strain described
above, and the cells were plated on galactose.
What did the investigators hope to see? The hope was that SNF1 and
SNF4 would interact, and in doing so would effectively make a complex
that was capable of activating expression from the GAL promoters, since it
now contained both the UAS binding region and the activator region of the
Gal4 protein, linked by the SNF1/SNF4 interaction. This should cause