Download The reverse two

Brief Introduction of Yeast Two-hybrid System
Xiaoqin Lai
Dec. 2, 2004
Dimensions of Information Complexity
Genomics vs. Post-Genomics
Genome:
30.000 genes
Transcriptome: 40-100.000 mRNAs
Proteome:
100-400.000 proteins
>1.000.000 interactions
Protein Interaction
106
Human Proteome
Transcripts
Human Genome
105
What is proteomics?
• Proteome: Expressed protein complement of a cell
• Proteomics:
– “The systematic study of the many and diverse properties of
proteins in a parallel manner with the aim of providing
detailed descriptions of the structure, function and control of
biological systems in health and disease”
Patterson & Aebersold Nat Genetics 33:S311 (2003)
– Just about everything that has something to do with proteins
and is “high-throughput” in some way
Proteomics tasks
a. Interaction mapping
b. Expression profiling
c. Activity profiling
d. Modification profiling
e. Localization profiling
Protein-Protein Interaction
 Protein-protein interactions are one of the most
important regulatory mechanisms in cells.
 Most of the cellular processes are coordinated by
specific protein interactions.
 By figuring the functions of non-annotated
proteins, biologist will better understand
molecular mechanism of biological events
Protein interaction analysis methods
Yeast-two hybrid
– Ito et al. PNAS (2000, 2001),
Uetz et al. Nature (2000)
(yeast)
– Giot et al. Science (2004)
(Drosophila)
– Li S., et al (2004) Science (C.
Elegans)
Biochemical purifications
In silico predictions
– Methods
Co-occurrence (Phylogenetic profiling)
Neighborhood (Operon)
Fusion (Rosetta)
– See review by Osterman and
Overbeek in Curr Opin Chem Biol.
(2003)
mRNA-co-expression
– Eisen et al., PNAS (1998) Marcotte
– Yeast
Nature (1999)
• Gavin et al. (2002) Nature
Synthetic lethals
• Ho et al. (2002) (HMS-PCI)
– Tong (2004)
Nature
• Krogan et al. (2004) Mol Others
Pull down, Far western,Co-IP
Cell
Protein Chip
Phage display
Fluorescence resonance energy transfer
Surface plasma resonance transfer
Reference databases
• Interactions
–
–
–
–
–
–
–
MIPS
DIP
YPD
Intact (EBI)
BIND/ Blueprint
GRID
MINT
• Prediction server
– Predictome (Boston U)
– Plex (UTexas)
– STRING (EMBL)
• Protein complexes
– MIPS
– YPD
GST pull-down assay
Yeast Two Hybrid Assay
Standard genetic screen for physical protein-protein
interactions
• Benefits:
– Simple
– Inexpensive
– Scalable and
automatable
Comparison of protein-protein
interaction screens
Differences between individual
methods and reference sets
Interaction density
energy production
aminoacid metabolism
other metabolism
translation
transcription
0
10
M
Interaction pro
1000 possible
P
T
transcriptional control
B
protein fate
F
cellular organization
O
transport and sensing
A
R
stress and defense
D
genome maintenance
C
cellular fate/organization
U
uncharacterized
Interaction density
E
G
EG
M
P
T
B
F
O
AR D
C
U
Functional biases
Comparison
Conclusions
The overlap between the individual
methods is surprisingly small
Different methods complement each other
Individual methods are not exhausted
Single experimental methods can be as
reliable as combined sets
Integration
[ Bader, G. and Hogue, C. (2002) Nat. Biot.]
[Kemmeren H., et al. (2002) Mol. Cell]
[Von Mering C., Krause, R., et al. (2002) Nature]
[Edwards et al. (2002) Trends Genet. ]
Number of citations in
Medline that describe
use of the yeast twohybrid system in 19891999 is growing
exponentially. Total
number of citations to
date approaches 3000.
Year
Yeast two-hybrid system:
a genetic assay for detecting protein-protein interactions
Suppose we have two proteins...
X
Y
and the question,
do these two protein bind each other?
One way of answering this question is to
use the yeast two hybrid method.
To understand the method we must first
consider how gene expression is regulated in yeast......
Yeast two-hybrid system:
a genetic assay for detecting protein-protein interactions
Regulation of gene expression in yeast
t
r
a
n
s
c
r
i
p
t
i
o
n
a
c
t
i
v
a
t
o
r
a
c
t
i
v
a
t
i
o
n
d
o
m
a
i
n
t
r
a
n
s
c
r
i
p
t
i
o
n
m
a
c
h
i
n
e
r
y
D
N
A
b
i
n
d
i
n
g
d
o
m
a
i
n
U
A
S
(
u
p
s
t
r
e
a
m
a
c
t
i
v
a
t
i
o
n
s
e
q
u
e
n
c
e
)
g
e
n
e
and now back to the question...
Yeast two-hybrid system:
a genetic assay for detecting protein-protein interactions
Regulation of gene expression in yeast
t
r
a
n
s
c
r
i
p
t
i
o
n
a
c
t
i
v
a
t
o
r
a
c
t
i
v
a
t
i
o
n
d
o
m
a
i
n
D
N
A
b
i
n
d
i
n
g
d
o
m
a
i
n
U
A
S
(
u
p
s
t
r
e
a
m
a
c
t
i
v
a
t
i
o
n
s
e
q
u
e
n
c
e
)
t
r
a
n
s
c
r
i
p
t
i
o
n
m
a
c
h
i
n
e
r
y
D
othese
tw
oprotein
bind?
g
e
n
e
Y
X
We start with yeast possessing a reporter gene (a gene
making a product that is easy to detect).
And now we introduce genes coding for two hybrid
proteins.....
r
e
p
o
r
t
e
r
g
e
n
e
Yeast two-hybrid system:
a genetic assay for detecting protein-protein interactions
Regulation of gene expression in yeast
t
r
a
n
s
c
r
i
p
t
i
o
n
a
c
t
i
v
a
t
o
r
a
c
t
i
v
a
t
i
o
n
d
o
m
a
i
n
t
r
a
n
s
c
r
i
p
t
i
o
n
m
a
c
h
i
n
e
r
y
D
N
A
b
i
n
d
i
n
g
d
o
m
a
i
n
g
e
n
e
U
A
S
(
u
p
s
t
r
e
a
m
a
c
t
i
v
a
t
i
o
n
s
e
q
u
e
n
c
e
)
Y
o
u
r
t
w
o
h
y
b
r
i
d
p
r
o
t
e
i
n
s
b
i
n
d
!
yes, we have expression of
the reporter indicating that
the proteins bind!
Y
t
h
u
s
f
o
r
m
i
n
g
a
f
u
n
c
t
i
o
n
a
l
t
r
a
n
s
c
r
i
p
t
i
o
n
a
c
t
i
v
a
t
o
r
.
.
. X
Y
X
X
X
Do these
proteins
bind?
Y
do we get expression of the reporter?
r
e
p
o
r
t
e
r
g
e
n
e
酵母双杂交系统
常用的DNA结合结构域:
GAL4（1-147）
LexA（E.coli 转录抑制因子）
常用的转录激活结构:
GAL4（768-881）
B42（ E.coli ）
VP16（疱疹病毒）
工作原理
酵母或大肠杆菌的转录因子(Gal4、LexA)的DNA结构域可以
将一个与其融合的蛋白质分子X(诱饵)带至报告基因的上游激
活序列(UAS)，并与之结合；
与转录因子的转录激活结构域(来自Gal4或VP16)融合的蛋白
质分子Y(靶蛋白)，可通过其与X蛋白质的相互作用，将激活结
构域带至报告基因的调控区；
DNA结合结构域和转录激活结构域在空间上的靠近，重建了
转录因子的功能，激活了下游报告基因的表达，表现为酵母可
以在特定的缺省培养基上生长，或在有底物X-gal时，形成蓝
色菌落
三个基本组成部分
表达诱饵蛋白的载体，诱饵即我们感兴趣的蛋白，
它和DNA结合结构域融合。
表达靶蛋白的载体，靶蛋白可以是一个已知的蛋
白，也可以是cDNA或基因组文库编码的蛋白。靶蛋
白和转录激活结构域融合。
一个或多个报告基因(如控制氨基酸合成的基因、
大肠杆菌的lacZ基因等)，位于DNA结合结构域识别
的调控区的下游。
Fishing with the yeast two-hybrid system
b
a
it
p
r
e
d
a
t
o
r
X
Y
d
o
e
s
X
b
i
n
d
w
i
t
h
a
p
r
o
t
e
i
n
?
in other words is there a protein Y?
To find out we are going to go fishing with the two-hybrid system.
W
e
w
i
l
l
u
s
e
X
a
s
b
a
i
t
.
.
.
.
t
o
t
r
y
t
o
c
a
t
c
h
Y
.
As reference in this description, here is how
the yeast two-hybrid expression system works.
bait
X
Y
transcription machinery
prey
lac 2
-galactosidase
X-gal
blue color
Fishing with the yeast two-hybrid system
b
a
it
b
a
i
t
p
r
e
d
a
t
o
r
X
p
r
o
t
e
i
n
X
Y
c
D
N
A
f
o
r
X
DNAbinding
domain
d
o
e
s
X
b
i
n
d
w
i
t
h
a
p
r
o
t
e
i
n
?
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
t
r
a
n
s
f
o
r
m
e
d
y
e
a
s
t
X
We'll start by making transformed yeast expressing X
bait
X
Y
transcription machinery
prey
lac 2
-galactosidase
X-gal
blue color
Fishing with the yeast two-hybrid system
b
a
it
b
a
i
t
p
r
o
t
e
i
n
X
c
D
N
A
f
o
r
X
DNAbinding
domain
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
p
r
e
d
a
t
o
r
p
r
e
d
a
t
o
r
X
Y
u
n
k
n
o
w
t
i
s
s
u
e
d
o
e
s
X
b
i
n
d
w
i
t
h
a
p
r
o
t
e
i
n
?
t
o
t
a
l
m
R
N
A
r
e
v
e
r
s
e
t
r
a
n
s
c
r
i
p
t
a
s
e
c
D
N
A
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
t
r
a
n
s
f
o
r
m
e
d
y
e
a
s
t
X
a
c
tiv
a
tio
n
d
o
m
a
in
bait
X
Y
transcription machinery
prey
lac 2
-galactosidase
X-gal
blue color
Fishing with the yeast two-hybrid system
b
a
it
b
a
i
t
p
r
o
t
e
i
n
X
c
D
N
A
f
o
r
X
DNAbinding
domain
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
p
r
e
d
a
t
o
r
p
r
e
d
a
t
o
r
X
Y
u
n
k
n
o
w
t
i
s
s
u
e
d
o
e
s
X
b
i
n
d
w
i
t
h
a
p
r
o
t
e
i
n
?
t
o
t
a
l
m
R
N
A
r
e
v
e
r
s
e
t
r
a
n
s
c
r
i
p
t
a
s
e
c
D
N
A
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
t
r
a
n
s
f
o
r
m
e
d
y
e
a
s
t
a
c
tiv
a
tio
n
d
o
m
a
in
X
e
x
t
r
a
c
t
p
l
a
s
m
i
d
s
bait
X
r
e
t
r
a
n
s
f
o
r
m
e
d
y
e
a
s
t
Y
transcription machinery
prey
lac 2
Now we make re-transformed yeast.
-galactosidase
X
Y
?
X-gal
blue color
Fishing with the yeast two-hybrid system
b
a
it
b
a
i
t
p
r
o
t
e
i
n
X
c
D
N
A
f
o
r
X
DNAbinding
domain
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
p
r
e
d
a
t
o
r
p
r
e
d
a
t
o
r
X
Y
u
n
k
n
o
w
t
i
s
s
u
e
d
o
e
s
X
b
i
n
d
w
i
t
h
a
p
r
o
t
e
i
n
?
t
o
t
a
l
m
R
N
A
r
e
v
e
r
s
e
t
r
a
n
s
c
r
i
p
t
a
s
e
c
D
N
A
y
e
a
s
t
p
l
a
s
m
i
d
e
x
p
r
e
s
s
i
o
n
v
e
c
t
o
r
t
r
a
n
s
f
o
r
m
e
d
y
e
a
s
t
a
c
tiv
a
tio
n
d
o
m
a
in
X
e
x
t
r
a
c
t
p
l
a
s
m
i
d
s
p
o
s
i
t
i
v
e
y
e
a
s
t
c
o
n
t
a
i
n
i
n
g
b
a
i
tp
l
u
s
p
r
e
d
a
t
o
r
!
r
e
t
r
a
n
s
f
o
r
m
e
d
y
e
a
s
t
bait
X
Y
transcription machinery
prey
a
g
a
r
p
l
a
t
e
lac 2
t
h
e
f
is
h
in
g
w
a
s
g
o
o
d
!
-galactosidase
X
Y
?
X-gal
blue color
Applications of YTH
高灵敏度地检测蛋白－蛋白的相互作用
确定蛋白相互作用的结构域或重要活性位点
寻找与靶蛋白相互作用的新蛋白
寻找具有药物治疗作用的小分子肽
寻找控制蛋白相互作用的化合物
蛋白相互作用图谱的绘制
Sources of yeast two-hybrid system
Matchmaker Yeast Two-hybrid System 3
DNA-BD : Amino acids 1-147 of the yeast Gal4 protein
binding to the Gal UAS upsream of the report genes
AD : Amino acids 768-881 of the Gal4 protein
a transcriptional activator
Reporter constructs in yeast strains AH109 and Y187
Sequence of the Gal4 DNA-BD recognition sites
MATCHMAKER Yeast Two-hybrid System 3 Vectors
Overview of performing a yeast two-hybrid screen
Interactions can be screened for strength
3-AT: Competitive Inhibitor of HIS3 protein
Yeast Two Hybrid Assay
• Y2H screens are notorious for a high level of false positives!
• Some baits activate transcription alone
• Some interactions occur by chance or due to incorrectly
folded proteins
• Some proteins are just ‘sticky’
Uetz, 2001
Protein interactions indicated by a YTH screen
False positives should be eliminated
Step 1: double screens avoid false positives
Step 2: independent validation of interaction
Pull-down assays (affinity isolation)
Co-localization of protein expression
Verification of putative positive clones
Verification of putative positive clones (con’d)
Yeast mating to verify protein interactions
Genome Scale Yeast Two Hybrid Assay
Red: positive screen 1
Green: positive screen 2
Yellow: positive for both
Uetz, 2001
Ito et al, 2001
Interactions
in the yeast
proteome
EXAMPLE
Troubleshooting Guide
Problem
BD/bait acitvates
reporter genes
Cause
The bait protein has
a transcriptional
activation domain.
This is especially
likely if the bait
protein is a
transcription factor.
Solution
If two test proteins are
being assayed,
switch from the BD to
the AD vector
and vice versa.
Remove the activating
domain by creating
specific deletions
within the gene.
Retest the deletion
constructs for
activation.
Problem
Excessive
background
Cause
Improper media
Resuspension of
transformed cells in
YPDA is too rich
Solution
Remake medium.
Add the appropriate
amount of 3-AT
Use water or TE.
Problem
Low transformation
efficiency
Cause
Solution
Improper media
Remake media
Cotransformation
Switch to sequential
transformation
The AD/library or
bait plasmid
transformation
Use more
Bait is toxi or
inhibiting to
transformation
Switch to a low
expressing BD vector
Repufy DNA
Problem
Failure to detect
known protein
interactions
Cause
Cell toxicity
Solution
Trunction
Use vectors that express
lower levels of the
fusion proteins
Low transforImprovement
Mation efficiency
Expression
Generate mutant forms
Folding
of the proteins
Location
The fused domain
occlude the site
of interaction
The development of yeast two-hybrid system
The yeast one-hybrid system
The Grow'n'Glow GFP One-Hybrid kit isolates genes for proteins that
bind a specific DNA element of interest. In addition to finding novel
DNA-binding proteins, the one-hybrid system can be used to
investigate the bases and amino acids involved in specific DNAprotein interactions. Proteins can be found that bind to any short DNA
element of interest.
The reverse two-hybrid system (1)
Reporter 1 (HIS3) is used for positive selection, while reporter 2
(URA3) is used for counterselection. Interaction between DBDand AD-fused proteins results in growth on medium lacking
histidine, but lethality on medium containing 5-fluoroorotic acid
(5FOA), a toxic metabolite of the URA pathway.
The reverse two-hybrid system (2)
Following mutagenesis of DBD- or AD- fusion protein,
mutations which weaken the interaction will display slow
growth on both media, while mutations or truncations which
completely abrogate the interaction will result in moderate to
strong growth on 5FOA medium, but no growth on histidinemedium.
Dual Bait design
Single prey with two different baits. Can be used to select preys that interact with the
DBD1-B but not DBD2-C from a library. Alternatively, if starting with a prey that
interacts with both DBD1-B and DBD2-C, it can be used to select for mutations or
molecules that selectively disrupt the interaction with one of the two baits.
A yeast tribrid system to characterize
protein-RNA interactions
Thanks!