Gene Cloning

Gene Cloning in the Functional Analysis of Proteins 299
expression of the lacZ gene, which as you recall had been placed under the
control of the GAL promoter, and it should also enable the strains to now
grow on galactose, as the new complex could now activate expression from
the GAL promoters within the cell (including the endogenous promoter
driving the GAL gene). Both these predictions were met: even though the
two essential domains of the Gal4 protein were now on different proteins,
the fact that these two proteins interacted enabled them to come together
to activate gene expression.
This remarkable system allows a number of different experiments to be
done. First, it can be used to define the regions of two proteins that interact
with each other. In the first experiment described above, complete coding
regions for the two interacting partners were used. However, by
expressing smaller parts of the proteins and checking for lacZ activity, it
would be possible to map the domains on the two proteins that are
required for them to interact. Such experiments have indeed been done.
It is important to realize that the two-hybrid system is not foolproof and
can give rise to both “false positives” (i.e. an interaction is apparently seen
even though it does not exist or is not significant in vivo) and “false negatives”
(an interaction which does occur is not seen using this system), and
any results from the yeast system need to be verified using other methods,
but the overall principle is a very useful one.
Another application of the yeast two-hybrid system is to look for partners
to a particular protein, rather than investigating known partners in
more depth. In such a system, the protein for which partners are being
sought is referred to as the “bait” protein, and it is the gene for this protein
which is fused to the DNA sequence encoding the DNA binding domain of
the Gal4 protein, exactly as in the method described above. Next, a cDNA
library is made in such a way that the cDNAs are inserted downstream from
the activator domain, such that a fusion protein will be produced. The
library is then transformed into a yeast strain containing the “bait” plasmid.
Plasmids in the library that contain a protein or protein domain that
interacts with the bait protein will lead to the formation of an active Gal4,
which will hence activate expression from a GAL promoter. Frequently in
this kind of experiment, a GAL promoter is placed upstream of a his gene,
and the experiment is done in a his auxotroph, enabling selection of only
those clones that contain a protein that will bind to the bait. The entire procedure
is shown in Figure 10.10.
Q10.7. With such a system, what medium should be used to select for the
clones of interest?
Q10.8. Can you think of a modification to this method that would enable
you to identify all the proteins that bind to a particular DNA sequence?