Brief Introduction of Yeast Two-hybrid System

Brief Introduction of Yeast Two-hybrid System

Xiaoqin Lai

Dec. 2, 2004

Genome: 30.000 genes

Transcriptome: 40-100.000 mRNAs

Proteome: 100-400.000 proteins >1.000.000 interactions

Dimensions of Information ComplexityGenomics vs. Post-Genomics

Human Genome

Human Proteome

Transcripts

Protein Interaction

105

106

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– Yeast

• Gavin et al. (2002) Nature • Ho et al. (2002) (HMS-PC

I) Nature• Krogan et al. (2004) Mol

Cell

Protein Chip

In silico predictions– Methods Co-occurrence (Phylogenetic profiling) Neighborhood (Operon) Fusion (Rosetta)– See review by Osterman and Overbe

ek in Curr Opin Chem Biol. (2003)

mRNA-co-expression– Eisen et al., PNAS (1998) Marcotte N

ature (1999)

Synthetic lethals– Tong (2004)

Others Pull down, Far western,Co-IP 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

GST

-fusi

on p

rote

inG

ST a

lone

Yeast Two Hybrid Assay

• Benefits:

– Simple

– Inexpensive

– Scalable and automatable

Standard genetic screen for physical protein-protein interactions

Comparison of protein-protein interaction screens

Differences between individual methods and reference sets

EG M P T B F O A R D C U

EG

P

T

B

F

O

AR

D

M

C

U

energy production

aminoacid metabolism

other metabolism

translation

transcription

transcriptional control

protein fate

cellular organization

transport and sensing

stress and defense

genome maintenance

cellular fate/organization

uncharacterized

Interaction density

Interaction pro 1000 possible

0 10

Interaction density

Functional biases

Comparison

ConclusionsThe overlap between the individual methods is surprisingly s

mallDifferent methods complement each otherIndividual methods are not exhaustedSingle experimental methods can be as reliable as combined

setsIntegration

[ 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 two-hybrid system in 1989-1999 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...

One way of answering this question is touse the yeast two hybrid method.

To understand the method we must first consider how gene expression is regulated in yeast......

and the question,do these two protein bind each other?

Yeast two-hybrid system:a genetic assay for detecting protein-protein interactions

Regulation of gene expression in yeast

geneUAS(upstream activation sequence)

transcription activator

DNA binding domain

activation domaintranscription machinery

and now back to the question...

Yeast two-hybrid system:a genetic assay for detecting protein-protein interactions

Do thesetwo proteinbind?

Regulation of gene expression in yeast

geneUAS(upstream activation sequence)

transcription activator

DNA binding domain

activation domaintranscription machinery

reporter gene

We start with yeast possessing a reporter gene (a genem aking a product that is easy to detect).

And now we introduce genes coding for two hybridproteins.....

Yeast two-hybrid system:a genetic assay for detecting protein-protein interactions

Regulation of gene expression in yeast

geneUAS(upstream activation sequence)

transcription activator

DNA binding domain

activation domaintranscription machinery

reporter gene

X

Y

X Y

X Y do we get expression of the reporter?

yes, we have expression ofthe reporter indicating thatthe proteins bind!

Do theseproteinsbind?

our two hybridproteins bind!

thus form ing a functionaltranscription activator...

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）

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

工作原理

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。

三个基本组成部分

Fishing with the yeast two-hybrid system

X Y

does X bindwith a protein?

bait predator

transcription machineryX Y

bait

preylac 2

-galactosidase

X-gal blue color

in other words is there a protein Y?

To find out we are going to go fishing with the two-hybrid system.

We will use X as bait.... to try to catch Y.

As reference in this description, here is howthe yeast two-hybrid expression system works.

Fishing with the yeast two-hybrid system

X Y

does X bindwith a protein?

bait predator

transcription machineryX Y

bait

preylac 2

-galactosidase

X-gal blue color

protein Xbait

cD N A for X

yeast plasm idexpression vector DN A -

bindingdom ain

transformedyeast

X

W e'll start by m aking transform ed yeast expressing X

Fishing with the yeast two-hybrid system

X Y

does X bindwith a protein?

bait predator

transcription machineryX Y

bait

preylac 2

-galactosidase

X-gal blue color

protein Xbait

cD N A for X

yeast plasm idexpression vector DN A -

bindingdom ain

transformedyeast

X

unknowpredator

tissue

total mRNA

reverse transcriptase

cDNA

yeast plasm id expression vector

activationdom ain

Fishing with the yeast two-hybrid system

X Y

does X bindwith a protein?

bait predator

transcription machineryX Y

bait

preylac 2

-galactosidase

X-gal blue color

protein Xbait

cD N A for X

yeast plasm idexpression vector DN A -

bindingdom ain

transformedyeast

X

unknowpredator

tissue

total mRNA

reverse transcriptase

cDNA

yeast plasm id expression vector

extract plasm ids

re-transformedyeast

X Y

?

activationdom ain

Now we m ake re-transform ed yeast.

Fishing with the yeast two-hybrid system

X Y

does X bindwith a protein?

bait predator

transcription machineryX Y

bait

preylac 2

-galactosidase

X-gal blue color

protein Xbait

cD N A for X

yeast plasm idexpression vector DN A -

bindingdom ain

transformedyeast

X

unknowpredator

tissue

total mRNA

reverse transcriptase

cDNA

yeast plasm id expression vector

extract plasm ids

re-transformedyeast

X Y

?

agar p late

activationdom ain

positive yeastcontaining

bait plus predator!

th e fish ingw as g ood!

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 Y187Reporter 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 screenOverview of performing a yeast two-hybrid screen

Interactions can be screened for strength

3-AT: Competitive Inhibitor of HIS3 protein

Yeast Two Hybrid Assay

Uetz, 2001

• 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’

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

Uetz, 2001 Red: positive screen 1 Green: positive screen 2Yellow: positive for both

Ito et al, 2001

Interactions in the yeast proteome

EXAMPLE

Troubleshooting Guide

Problem Cause Solution

BD/bait acitvates reporter genes

The bait protein has a transcriptionalactivation domain. This is especiallylikely if the bait protein is a transcription factor.

If two test proteins are being assayed,switch from the BD to the AD vectorand vice versa.

Remove the activating domain by creatingspecific deletions within the gene.Retest the deletion constructs for activation.

Problem Cause Solution

Excessive background

Improper media Remake medium. Add the appropriateamount of 3-AT

Resuspension of transformed cells inYPDA is too rich

Use water or TE.

Problem Cause Solution

Low transformation efficiency

Improper media Remake media

Cotransformation Switch to sequentialtransformation

The AD/library or bait plasmid transformation

Use more

Repufy DNA

Bait is toxi or inhibiting to transformation

Switch to a low expressing BD vector

Problem Cause Solution

Failure to detectknown proteininteractions

Cell toxicity Trunction

Use vectors that expresslower levels of thefusion proteins

Low transfor-Mation efficiency

Improvement

ExpressionFoldingLocationThe fused domain occlude the siteof interaction

Generate mutant formsof the proteins

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 DNA-protein interactions. Proteins can be found that bind to any short DNA element of interest.

Reporter 1 (HIS3) is used for positive selection, while reporter 2 (URA3) is used for counterselection. Interaction between DBD- and 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 (1)

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 histidine- medium.

The reverse two-hybrid system (2)

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.

Dual Bait design

A yeast tribrid system to characterize protein-RNA interactions

Thanks!