22 59 Assaying ProteinProtein Interactions

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Chapter 5 / Molecular Tools for Studying Genes and Gene Activity
SUMMARY Footprinting is a means of finding the
target DNA sequence, or binding site, of a DNAbinding protein. DNase footprinting is performed
by binding the protein to its end-labeled DNA target, then attacking the DNA–protein complex with
DNase. When the resulting DNA fragments are
electrophoresed, the protein binding site shows up
as a gap, or “footprint” in the pattern where the
protein protected the DNA from degradation.
DMS footprinting follows a similar principle, except that the DNA methylating agent DMS, instead
of DNase, is used to attack the DNA– protein complex. The DNA is then broken at the methylated
sites. Unmethylated (or hypermethylated) sites
show up on electrophoresis of the labeled DNA
fragments and demonstrate where the protein
bound to the DNA. Hydroxyl radical footprinting
uses copper- or iron-containing organometallic
complexes to generate hydroxyl radicals that break
DNA strands.
A
Bead
(a)
Immunoprecipitate
DNA–protein complex.
(b)
Identify DNA by PCR.
n
Chromatin Immunoprecipitation (ChIP)
Chromatin immunoprecipitation (ChIP) is a way of discovering whether a given protein is bound to a given gene
in chromatin—the DNA–protein complex that is the natural state of DNA in a living cell (Chapter 13). Figure 5.39
illustrates the method. One starts with chromatin isolated from cells and adds formaldehyde to form covalent
bonds between DNA and any proteins bound to it. Then
one shears the chromatin by sonication to produce short,
double-stranded DNA fragments cross-linked to proteins.
Next, one makes cell extracts and immunoprecipitates the
protein–DNA complexes with antibodies directed against
a protein of interest, as described earlier in this chapter.
This precipitates that specific protein, and the DNA to
which it binds. To see if that DNA contains the gene
of interest, one performs PCR (Chapter 4) on the immunoprecipitate with primers designed to amplify that gene.
If the gene is present, a DNA fragment of predictable
size will result and be detectable as a band after gel
electrophoresis.
SUMMARY Chromatin immunoprecipitation de-
tects a specific protein–DNA interaction in chromatin in vivo. It uses an antibody to precipitate a
particular protein in complex with DNA, and PCR
to determine whether the protein binds near a particular gene.
Figure 5.39 Chromatin immunoprecipitation. The chromatin has
already been cross-linked with formaldehyde and sheared into short
pieces. (a) The immunoprecipitation step. An antibody (red) has
bound to an epitope (yellow) attached to a protein of interest
(purple), which in turn is bound to a specific site on a doublestranded DNA (blue). The antibody is bound to staphylococcal
protein A (or G), which is coupled to a large bead that can be easily
purified by centrifugation. The bead can even be magnetic, so the
immune complexes can be drawn to the bottom of a tube with a
magnet. The antibody does not bind to the other proteins (green and
orange) to which the epitope is not attached. (b) Identifying the DNA
in the immunoprecipitate. Primers specific for the DNA of interest are
used in a PCR reaction to amplify a portion of the DNA. Production
of a DNA fragment of the correct predicted size indicates that the
protein did indeed bind to the DNA of interest. (The primers do not
amplify the exact sequence to which the protein binds,but an
adjacent portion of the gene of interest.)
5.9
Assaying Protein–Protein
Interactions
Protein–protein interactions are also extremely important
in molecular biology, and there are a number of ways to assay them. Immunoprecipitation, which we discussed earlier
in this chapter, is one way: If an antibody directed against a
particular protein (X) precipitates both proteins X and Y
together, but has no affinity for protein Y on its own, it is
very likely that protein Y associates with protein X.
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5.9 Assaying Protein–Protein Interactions
Another popular method is called a yeast two-hybrid assay.
Figure 5.40 describes a generic version of this very sensitive
technique, which is designed to demonstrate binding—even
transient binding—between two proteins. The yeast twohybrid assay takes advantage of two facts, discussed in
Chapter 12: (1) that transcription activators typically have a
DNA-binding domain and a transcription-activating
domain; and (2) that these two domains have self-contained
activities. To assay for binding between two proteins, X and
Y, one can arrange for yeast cells to express these two
proteins as fusion proteins, pictured in Figure 5.40b. Protein
X is fused to a DNA-binding domain, and protein Y is fused
to a transcription-activating domain. Now if proteins X and
Y interact, that brings the DNA-binding and transcriptionactivating domains together, and activates transcription of a
reporter gene (typically lacZ).
(a) Standard activation
Basal complex
IacZ
(b) Two-hybrid activation
X
Y
AD
BD
Transcription
Basal complex
IacZ
(c) Two-hybrid screen
C
A
AD
AD
E
Z
BD
One can even use the yeast two-hybrid system to fish
for unknown proteins that interact with a known protein
(Z). In a screen such as Figure 5.40c, one would prepare
a library of cDNAs linked to the coding region for a
transcription-activating domain and express these hybrid
genes, along with a gene encoding the DNA-binding
domain—Z hybrid gene, in yeast cells. In practice, each
yeast cell would make a different fusion protein (AD–A,
AD–B, AD–C, etc.), along with the BD–Z fusion protein,
but they are all pictured here together for simplicity. We
can see that AD–D binds to BD–Z and activates transcription, but none of the other fusion proteins can do
this because they cannot interact with BD–Z. Once clones
that activate transcription are found, the plasmid bearing
the AD–D hybrid gene is isolated and the D portion is
sequenced to find out what it codes for. Because the yeast
two-hybrid assay is indirect, it is subject to artifacts.
Transcription
AD
BD
113
B
AD
D
AD
AD
Transcription
Basal complex
IacZ
Figure 5.40 Principle of the yeast two-hybrid assay. (a) Standard
model of transcription activation. The DNA-binding domain (BD, red)
of an activator binds to an enhancer (pink), and the activating
domain (AD green) interacts with the basal complex (orange),
recruiting it to the promoter (brown). This stimulates transcription.
(b) Two-hybrid assay for protein–protein interaction. Using gene
cloning techniques (Chapter 4), link the gene for one protein
(X, turquoise) to the part of a gene encoding a DNA-binding domain
to encode one hybrid protein; link the gene for another protein
(Y, yellow) to the part of a gene encoding a transcription-activating
domain to encode a second hybrid protein. When plasmids encoding
these two hybrid proteins are introduced into yeast cells bearing the
appropriate promoter, enhancer, and reporter gene (lacZ, purple, in
this case), the two hybrid proteins can get together as shown to
serve as an activator. Activated transcription produces abundant
reporter gene product, which can be detected with a colorimetric
assay, using X-gal, for example. One hybrid protein contributes a
DNA-binding domain, and the other contributes a transcriptionactivating domain. The two parts of the activator are held together
by the interaction between proteins X and Y. If X and Y interact, and
X-gal is used in the assay for the reporter gene product, the yeast
cells will turn blue. If X and Y do not interact, no activator will form,
and no activation of the reporter gene will occur. In this case, the
yeast cells will remain white in the presence of X-gal. The GAL4
DNA-binding domain and transcription-activating domain are
traditionally used in this assay, but other possibilities exist.
(c) Two-hybrid screen for a protein that interacts with protein Z.
Yeast cells are transformed with two plasmids: one encoding a
DNA-binding domain (red) coupled to a “bait” protein (Z, turquoise).
The other is a set of plasmids containing many cDNAs coupled to
the coding region for a transcription-activating domain. Each of
these encodes a fusion protein containing the activating domain
(green) fused to an unknown cDNA product (the “prey”). Each yeast
cell is transformed with just one of these prey-encoding plasmids,
but several of their products are shown together here for convenience.
One prey protein (D, yellow) interacts with the bait protein, Z. This
brings together the DNA-binding domain and the transcriptionactivating domain so they can activate the reporter gene. Now the
experimenter can purify the prey plasmid from this positive clone and
thereby get an idea about the nature of the prey protein.