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