47 123 Independence of the Domains of Activators

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12.3 Independence of the Domains of Activators
may require the extra interaction of the leucine zippers to
ensure dimerization of the protein monomers.
SUMMARY The bZIP proteins dimerize through a
leucine zipper, which puts the adjacent basic regions
of each monomer in position to embrace the DNA
target site like a pair of tongs. Similarly, the bHLH
proteins dimerize through a helix-loop-helix motif,
which allows the basic parts of each long helix to
grasp the DNA target site, much as the bZIP proteins do. The bHLH and bHLH-ZIP domains bind
to DNA in the same way, but the latter have extra
dimerization potential due to their leucine zippers.
12.3 Independence of the
Domains of Activators
We have now seen several examples of DNA-binding and
transcription-activating domains in activators. These
domains are separated physically on the proteins, they fold
independently of each other to form distinct threedimensional structures, and they operate independently of
each other. Roger Brent and Mark Ptashne demonstrated
this independence by creating a chimeric factor with the
DNA-binding domain of one protein and the transcriptionactivating domain of the other. This hybrid protein functioned as an activator, with its specificity dictated by its
DNA-binding domain.
Brent and Ptashne started with the genes for two proteins: GAL4 and LexA. We have already studied the DNAbinding and transcription-activating domains of GAL4;
LexA is a prokaryotic repressor that binds to lexA operators and represses downstream genes in E. coli cells. It
does not normally have a transcription-activating domain,
because that is not its function. By cutting and recombining fragments of the two genes, Brent and Ptashne created
a chimeric gene containing the coding regions for the
transcription-activating domain of GAL4 and the DNAbinding domain of LexA. To assay the activity of the
protein product of this gene, they introduced two plasmids
into yeast cells. The first plasmid had the chimeric gene,
which produced its hybrid product. The second contained
a promoter responsive to GAL4 (either the GAL1 or the
CYC1 promoter), linked to the E. coli b-galactosidase
gene, which served as a reporter gene (Chapter 5). The
more transcription from the GAL4-responsive promoter,
the more b-galactosidase was produced. Therefore, by assaying for b-galactosidase, Brent and Ptashne could determine the transcription rate.
One more element was necessary to make this assay
work: a binding site for the chimeric protein. The normal
binding site for GAL4 is an upstream enhancer called UASG.
However, this site would not be recognized by the chimeric
protein, which has a LexA DNA-binding domain. To make
the GAL1 promoter responsive to activation, the investigators had to introduce a DNA target for the LexA DNAbinding domain. Therefore, they inserted a lexA operator in
place of UASG. It is important to note that a lexA operator
would not normally be found in a yeast cell; it was placed
there just for the purpose of this experiment. Now the question is: Did the chimeric protein activate the GAL1 gene?
The answer is yes, as Figure 12.12 demonstrates. The
three test plasmids contained UASG, no target site, or the
lexA operator. The activator was either LexA-GAL4, as we
have discussed, or LexA (a negative control). With UASG
present (Figure 12.12a), a great deal of b-galactosidase
was made, regardless of which activator was present. This
is because the yeast cells themselves make GAL4, which
can activate via UASG. When no DNA target site was present
(Figure 12.12b), no b-galactosidase could be made. Finally,
when the lexA operator replaced UASG (Figure 12.12c), the
LexA-GAL4 chimeric protein could activate b-galactosidase
production over 500-fold. Thus, one can replace the
β-galactosidase
(units)
(a)
GAL4
UASG
GAL1
lacZ
1800
GAL1
lacZ
0
GAL1
lacZ
520
(b)
(c)
LexA-GAL4
lexA op.
Figure 12.12 Activity of a chimeric transcription factor. Brent and
Ptashne introduced two plasmids into yeast cells: (1) a plasmid
encoding LexA-GAL4, a hybrid protein containing the transcriptionactivating domain of GAL4 (green) and the DNA-binding domain of LexA
(blue); and (2) one of the test plasmid constructs shown in panels a–c.
Each of the test plasmids had the GAL1 promoter linked to a reporter
gene (the E. coli lacZ gene). The chimeric protein LexA-GAL4 was used
as the activator. The production of b-galactosidase (given at right) is a
measure of promoter activity. (a) With a UASG element, transcription
was very active and did not depend on the added transcription factor,
because endogenous GAL4 could activate via UASG. (b) With no DNA
target site, LexA-GAL4 could not activate, because it could not bind
to the DNA near the GAL1 promoter. (c) With the lexA operator,
transcription was greatly stimulated by the LexA-GAL4 chimeric factor.
The LexA DNA-binding domain could bind to the lexA operator, and
the GAL4 transcription-activating domain could enhance transcription
from the GAL1 promoter.