Note: the following is useful for background information. Please see Protocols Page for more updated, and recommended interactor hunt protocols.

Interaction trap cloning with yeast.

The following manuscript is a draft of a chapter appearing in "DNA Cloning 2: A Practical Approach, Expression Systems", 2nd Edition, eds D. M. Glover and B. D. Hames. Oxford University Press, 1995.
 
 

Russell L. Finley Jr. and Roger Brent

Department of Molecular Biology
Massachusetts General Hospital
and Department of Genetics
Harvard Medical School
Boston Massachusetts 02114
U.S.A.
 
 

Address:
Department of Molecular Biology
Massachusetts General Hospital
50 Blossom St.
Boston Massachusetts 02114
USA

Phone (617) 726-5925
FAX (617) 726-6893
e-mail brent(at)frodo.mgh.harvard.edu
finley(at)frodo.mgh.harvard,edu
 
 

Contents

1. Introduction
1.1 Background
1.2 The interaction trap
2. Making and testing baits
2.1 LexA fusion expression plasmids
2.2 Reporters and yeast strains
2.2.1 LEU2 reporter strains
2.2.2 lacZ reporters
2.3 Testing the bait protein
2.3.1 Testing whether the bait protein activates transcription of the
reporters

Protocol 1. Testing baits for transcription activation

2.3.2 Demonstrating that the bait enters the yeast nucleus and binds
operators
Protocol 2. The repression assay
2.3.3 Verifying that a full-length fusion protein is made.

3. Libraries
4. An interactor hunt
4.1 Introducing the library into the selection strain.
4.1.1 Selecting interactors from library transformants.
4.1.2 Performing a one step selection for interactors

Protocol 3. Transforming the selection strain with library DNA.

4.2 Isolating yeast with galactose dependent Leu+ and lacZ+
phenotypes

Protocol 4. Selecting interactors.

5. Verifying specificity

Protocol 5. Isolating and classifying library plasmids.
Protocol 6. Determining specificity of interactors.

6. Using a mating assay to verify specificity.
Figure 4a. Mating assay cartoon.
Figure 4b. Mating assay result.

Protocol 7. Mating assay.

7. Expected results.

Appendix
Sequencing and PCR primers for pEG202 and pJG4-5
Media recipes.
 
 
 

Interaction trap cloning with yeast.
 

1. Introduction
The interaction trap is a two-hybrid system for cloning cDNAs
that encode proteins that interact with a protein whose coding
sequences are known. The method uses the transcription of yeast
reporter genes as a synthetic phenotype to detect protein-protein
interactions. It can also be used to study interactions between
known proteins.

1.1 Background

The two-hybrid approach takes advantage of the modular
domain structure of eukaryotic transcription factors. Many
eukaryotic transcription activators have at least two distinct
functional domains, one that directs binding to specific DNA
sequences and one that activates transcription (1, 2). This modular
structure is best illustrated by yeast experiments showing that the
DNA-binding domains or activation domains can be exchanged from
one transcription factor to the next and retain function. For example,
when the DNA-binding domain of the yeast transcription factor Gal4
is replaced with the DNA binding domain of the bacterial repressor
LexA, the resulting hybrid protein activates transcription of genes
containing upstream LexA binding sites (3). Similarly, when the DNA
binding domain of Gal4, which by itself does not activate
transcription, is fused to activation domains from other proteins the
resulting hybrid proteins activate transcription of reporters with
upstream Gal4 binding sites (4-6). A crucial corollary of the modular
nature of transcription activators is that the DNA-binding and
activation domains need not be covalently attached to each other for
activation to occur. This was first demonstrated by Ma and Ptashne
(7) with a Gal4 derivative that contained the DNA-binding domain as
well as a domain that interacts with another yeast protein, Gal80, but
that lacked the activation domain. When this derivative was
expressed in yeast it did not activate transcription of a reporter gene
containing upstream Gal4 binding sites. However, when it was co-
expressed with a second, hybrid protein, consisting of Gal80 fused to
an activation domain, interaction between the Gal4 DNA-binding
derivative and the Gal80-activation domain hybrid resulted in
activation of the reporter gene.

The general utility of the modularity of transcription factors
was demonstrated by Fields and Song (8) who showed that yeast
transcription could be used to assay the interaction between two
proteins if one of them was fused to a DNA-binding domain and the
other was fused to an activation domain. In their experiment, one of
the hybrid proteins contained the DNA-binding domain of Gal4 fused
to the yeast protein Snf1, and the other contained the activation
domain of Gal4 fused to another yeast protein, Snf4. When Snf1 and
Snf4 interacted they brought together the DNA-binding and
activation domains, so that the two hybrid proteins bound to Gal4
binding sites upstream of a lacZ reporter gene and activated its
transcription. Thus, the interaction between Snf1 and Snf4 was
assayed as production of beta-galactosidase. The success of this
experiment prompted Fields and Song to make the seminal
suggestion that yeast transcription could be used in this way to clone
cDNAs encoding proteins that interact with a given known protein
(8). In their scheme, a known protein is expressed fused to the DNA-
binding domain of Gal4, and a cDNA library is expressed so that
proteins encoded by the cDNA are fused to an activation domain
(activation-tagged). Transcription of a reporter gene will be
activated in yeast containing activation-tagged cDNA-encoded
proteins that interact with the known protein.

Based on this suggestion, two-hybrid cloning systems have
been developed in several labs (9-13). All have three basic
components: Yeast vectors for expression of a known protein fused to
a DNA-binding domain, yeast vectors that direct expression of cDNA-
encoded proteins fused to a transcription activation domain, and
yeast reporter genes that contain binding sites for the DNA-binding
domain. These components differ in detail from one system to the
other. All systems utilize the DNA binding domain from either Gal4
or LexA. The Gal4 domain is efficiently localized to the yeast nucleus
where it binds with high affinity to well-defined binding sites which
can be placed upstream of reporter genes (14-16). LexA does not
have a nuclear localization signal, but enters the yeast nucleus and,
when expressed at a sufficient level, efficiently occupies LexA
binding sites (operators) placed upstream of a reporter gene (3, 17,
18). No endogenous yeast proteins bind to the LexA operators.
Different systems also utilize different reporters. Most systems use a
reporter that has a yeast promoter, either from the GAL1 gene or the
CYC1 gene, fused to lacZ (19, 20). These lacZ fusions either reside on
multicopy yeast plasmids or are integrated into a yeast chromosome.
To make the lacZ fusions into appropriate reporters, the GAL1 or
CYC1 transcription regulatory regions have been removed and
replaced with binding sites that are recognized by the DNA-binding
domain being used. A screen for activation of the lacZ reporters is
performed by plating yeast on indicator plates that contain X-Gal (5-
bromo-4-chloro-3-indolyl-b-D-galactoside); on this medium yeast in
which the reporters are transcribed produce beta-galactosidase and turn
blue. Some systems use a second reporter gene and a yeast strain
that requires expression of this reporter to grow on a particular
medium. These "selectable marker" genes usually encode enzymes
required for the biosynthesis of an amino acid. Such reporters have
the marked advantage of providing a selection for cDNAs that encode
interacting proteins, rather than a visual screen for blue yeast. To
make appropriate reporters from the marker genes their upstream
transcription regulatory elements have been replaced by binding
sites for a DNA-binding domain. The HIS3 and LEU2 genes have both
been used as reporters in conjunction with appropriate yeast strains
that require their expression to grow on media lacking either
histidine or leucine, respectively.

Finally, different systems use different means to express
activation-tagged cDNA proteins. In all current schemes the cDNA-
encoded proteins are expressed with an activation domain at the
amino terminus. The activation domains used include the strong
activation domain from Gal4, the very strong activation domain from
the Herpes simplex virus protein VP16, or a weaker activation
domain derived from bacteria, called B42. The activation-tagged
cDNA-encoded proteins are expressed either from a constitutive
promoter, or from a conditional promoter such as that of the GAL1
gene. Use of a conditional promoter makes it possible to quickly
demonstrate that activation of the reporter gene is dependent on
expression of the activation-tagged cDNA proteins.

Many of these systems now provide the investigator with a
relatively good chance to recover proteins that interact with other
proteins. Because most are based on the same concepts, some of their
components are often interchangeable. However, different systems
utilize the yeast selectable markers in different ways. Moreover,
systems that employ the DNA-binding domain of Gal4 must use a
yeast strain that lacks wild type Gal4; these system cannot use
library vectors that direct synthesis of the activation-tagged proteins
from the GAL1 promoter whose transcription requires Gal4.

1.2 The interaction trap

The interaction trap is an implementation of the two-hybrid
system developed by Gyuris et. al (11). It consists of three critical
components (see Figure 1). First, it uses a vector for expression of a
protein of interest fused to LexA. Because the goal of interaction
trap cloning is to find proteins that interact with the protein fused to
LexA, this hybrid is referred to as the "bait". Second, the trap uses a
yeast strain with two reporter genes. One reporter is a yeast LEU2
derivative that has its normal upstream regulatory sequences
replaced with LexA operators. Transcription of the LexA-operator-
LEU2 gene (LexAop-LEU2) can be measured by the ability of the
strain to grow in the absence of leucine, which requires the LEU2
gene product. The LexAop-LEU2 gene is integrated into the yeast
chromosome. The other reporter gene is lacZ, which provides a
secondary assay of activation by the bait and activation-tagged
proteins interacting with it, as well as some quantitative information
about the interaction. Third, the interaction trap uses a library
plasmid that directs the conditional expression of cDNA-encoded
proteins fused at their amino termini to a moiety containing three
domains: a nuclear localization signal, a transcription activation
domain, and an epitope tag. The activation-tagged cDNA-encoded
protein is expressed from the yeast GAL1 promoter, which is induced
by galactose and repressed by glucose.

The interaction trap is illustrated in Figure 1. The bait protein
is constitutively expressed. It binds to LexA operators upstream of
the reporter genes LEU2 and lacZ but does not activate their
transcription. The activation-tagged cDNA-encoded protein is
conditionally expressed from the GAL1 promoter. In glucose
medium the GAL1 promoter is repressed, no cDNA-encoded protein
is made, and the yeast does not grow in the absence of leucine.
When the yeast are grown on galactose medium, activation-tagged
cDNA-encoded proteins are expressed, and those that interact with
the bait activate transcription of the LEU2 and lacZ reporters. Thus,
cells containing activation-tagged cDNA proteins that interact with
the bait form colonies on galactose medium lacking leucine and form
blue colonies on galactose X-Gal plates.

(Figure 1. The interaction trap)

An outline of an interactor hunt is presented in Figure 2. The
protocols for using the interaction trap described below require
knowledge of a few basic yeast microbiological and genetic
techniques. A more detailed description of such techniques, together
with recipes for appropriate media can be found elsewhere (21-23).

(Figure 2. Flow chart of an interactor hunt.)

2. Making and testing baits

2.1 LexA fusion expression plasmids

To make a plasmid that directs the synthesis of the LexA fusion
or "bait" protein, the coding region for the protein of interest is
inserted into pEG202 or a related plasmid (11) (see Appendix).
pEG202 is a multicopy yeast plasmid containing the yeast 2 mm
origin of replication and the selectable marker gene HIS3, as well as
the full-length LexA coding region flanked by the yeast ADH1
promoter and terminator. Bait proteins expressed from this plasmid
contain amino acids 1 to 202 of LexA, which include the DNA-binding
and dimerization domains. Downstream of the LexA coding region in
pEG202 are unique EcoRI, BamHI, SalI, NcoI, NotI, and XhoI cloning
sites. The bait plasmid can be introduced and maintained in a his3
yeast strain (e.g. EGY48, see below) by selecting transformants on
media lacking histidine. Transformants will constitutively express
the protein of interest with LexA at its amino terminus. Although it
does not contain a yeast nuclear localization signal, LexA and most