Twohybrid approaches to finding proteinprotein interactions

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Two-hybrid approaches to finding protein-protein
interactions
Yeast two-hybrid -based on the fact that
transcription factors such as yeast GAL4 have
independently operating transcriptional
activation and DNA-binding domains that can
be separated from each other -bait protein is
fused to a DNA-binding domain (BD) and screened
against a cDNA library in which cDNAs
are expressed as fusion proteins with an
activation domain (AD) -when binding occurs
between bait and library protein, a
functional transcription factor is formed
that can turn on reporter genes with
the appropriate target sequences in
their promoter-enhancer region -activation
domains used in yeast two-hybrid
screening -GAL4 -viral protein VP16 -acidic
bacterial peptide B42
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-several variations of the yeast two-hybrid
system have been developed, in the lab we will be
using the interaction trap method -in this
system, the bait protein is fused to the BD of
the bacterial repressor LexA, while the library
proteins are fused to AD of B42 -reporter genes
contain LexA operator sequences and will be
turned on by the chimaeric GAL4-LexA factor when
protein-protein interactions occur between bait
and library -yeast host strain in this system is
EGY48 which has the genotype a, his3, trp1,
ura3-52, lex(leu2)3a -bait is cloned into
pEG202 where it is fused to BD of LexA -pEG202
is selected for by growth on his-
medium -library is cloned into pJG4-5 where
cDNAs can be expressed fused to B42 -pJG4-5 is
selected for by growth on trp- medium
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-numerous cDNA libraries have been created that
will work in the interaction trap system -lacZ
reporter gene under control of lexA operator is
present in plasmid pSH18, which is selected for
by growth on ura- media -two-hybrid
interactions are detected through the expression
of the two reporter genes leu2 and
lacZ -expression of leu2 is selected for by
growth on leu- medium, while lacZ is detected
by growth on X-gal
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Variations on the yeast two-hybrid
technique Three-hybrid method for detecting
RNA-protein interactions - two-hybrid-derived
method has been developed that allows screening
for proteins interacting with a given RNA -this
system is commercially available from Invitrogen
as the RNA-Protein Hybrid Hunter kit -in this
system, the lexA BD is expressed as a fusion
with the coat protein of the MS2 bacteriophage
using the vector pHybLex/Zeo- MS2 -the RNA of
interest (i.e. the bait RNA) is expressed as a
fusion with MS2 RNA by cloning the DNA encoding
it into the vector pRH3 or pRH5 -finally, the
cDNAs encoding the potential prey proteins are
included in a vector that will express them as
fusions with an AD, for example that from VP16 or
B42 -all 3 vectors are introduced into the
L40-ura3 strain of S. cerevisiae and tested for
reporter gene expression
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-in this system the lexA operator-containing
reporter genes are HIS3 and lacZ -system is
based on the observation that the MS2 coat
protein binds to the MS2 RNA with high
specificity at a 33 nucleotide sequence that
forms a hairpin loop structure -reporter gene
expression is induced by joining the lexA BD to
the AD through a three-hybrid connector
consisting of (i) MS2 coat protein (ii)
MS2/bait RNA (iii) RNA-binding prey protein
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-efficacy of this method has been tested by
looking at two well known RNA-protein
interactions -recall that the iron response
element (IRE) is a stem-loop structure found in
the untranslated regions of mRNAs encoding
certain proteins involved in iron
utilization -for example, the protein
transferrin is involved in transporting iron into
the cell when cytoplasmic iron levels are high,
transferrin levels are low -this responsiveness
is controlled at the level of mRNA
stability -transferrin mRNA has IREs in its 3
UTR -when iron levels are low, an IRE
binding protein (IRP) binds to IREs in the
transferrin mRNA, probably by inhibiting the
function of destabilizing elements in the
vicinity -thus, when iron is low the transferrin
mRNA is stable and translated -when iron levels
are high, iron binds to IRP which then
dissociates from transferrin mRNA, exposing the
destabilizing elements and promoting degradation
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-tests with IRE and IRP demonstrate that lacZ
expression is only induced when all components of
the three-hybrid are present
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-another RNA-protein interaction used to test
the system is binding of the HIV Tat
transcription factor to the target TAR sequence
in the RNA product of a previous round of
transcription
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One-hybrid identification of DNA-protein
interactions -if one has a short DNA sequence
that is suspected to be a protein binding site, a
one-hybrid screen can be done -in this system,
the DNA sequence of interest (bait) is inserted
upstream of the minimal promoter of the reporter
gene as multiple tandem copies -cDNA library is,
as usual, expressed as fusion to AD -if
expressed protein binds to bait DNA, this will
result in functional transcription factor and
reporter gene expression
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A three-hybrid approach to investigate
interactions involving three proteins -the
pBridge vector replaces the usual BD bait
vector -with pBridge, one protein is fused to BD
and constitutively expressed, but another protein
of interest is expressed only in the absence of
methionine (can be switched on and off) -the
other protein is expressed from a conventional
two-hybrid vector as a fusion to an AD -effects
of bridging protein on interaction between
other two proteins can be tested by comparing
reporter gene expression with or without
induction of the bridging protein
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The bridging protein can contribute to the
interaction between the other two proteins in a
number of ways -link two proteins together by
binding both -stabilize a weak interaction
between two proteins -modify one or both
of the proteins -inhibit the two-hybrid
interaction
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Two-hybrid analysis in bacteria - a bacterial
two-hybrid system has been developed, based on
the observation that an arbitrary interaction
between a DNA-bound protein and bacterial RNA
polymerase can activate transcription -such an
interaction stabilizes the binding of RNA
polymerase close to promoter, and probably
acts similarly to the UP element in strongly
expressed bacterial genes -in the BacterioMatch
system marketed by Stratagene, the bait protein
is expressed as a fusion with the cI l
repressor protein -the ampr and lacZ reporter
genes have an upstream l operator sequence that
will bind the cI-bait fusion -the target
proteins from cDNA library are expressed as
fusions with the a subunit of RNA polymerase -if
bait interacts with target, we will get linking
of DNA-bound cI with a subunit of RNA polymerase
and enhanced transcription due to stronger
association with promoter
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AraC/LexA-based E. coli two-hybrid system -this
system based on the ara operon arabinose-
AraC protein acts as transcriptional activator
- arabinose- AraC causes DNA loop formation,
blocking access of RNA polymerase
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-the bait protein is fused to AraC, while the
prey is fused to LexA -in the absence of
interaction, reporter gene expression is turned
ON through AraC acting as an activator of
transcription -when there is interaction between
bait and prey, the promoter region is bent into
a repression loop, as the prey-LexA fusion
protein is also bound to LexA operator sequences
in the reporter gene promoter region -in this
system binding of bait to prey is
therefore visualized by REPRESSION of reporter
gene expression
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Mammalian two-hybrid assays -mammalian
two-hybrid analysis is generally used to validate
mammalian protein-protein interactions detected
in other systems -because the assay is
performed in mammalian cells, proteins are more
likely to be in a native confirmation and
experimental results are more likely to represent
biologically significant interactions -a kit
for doing mammalian two-hybrid is marketed by
Clontech -the reporter gene used is bacterial
chloramphenicol (CAM) acetyl transferase
(CAT) -CAT is one of the most heavily used
reporter genes used to measure eukaryotic
transcription -in one of the most popular CAT
assays , extracts from cells of interest are
mixed with radioactive CAM and an acetyl donor
(acetyl CoA) -thin layer chromatography is used
to separate CAM from its acetylated
products -the higher the promoter activity the
greater the concentration of acetylated
CAM -because eukaryotic cells lack endogenous
CAT, the background CAT level is zero
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REFERENCES Finley, R. L., Jr., and Brent, R.
(1995). Interaction trap cloning with yeast. In
DNA Cloning, Expression Systems A Practical
Approach, B. D. Hames and D. M. Glover, eds.
(Oxford Oxford University Press), pp.
169-203. Golemis et al. Current Protocols in
Molecular Biology, Supplement 46. Interaction
Trap/Two-Hybrid System to Identify Interacting
Proteins. Wiley. Hu JC, Kornacker MG, Hochschild
A. Escherichia coli one- and two-hybrid systems
for the analysis and identification of
protein-protein interactions. Methods. 2000
Jan20(1)80-94. Lewin B. Genes VII . Oxford
University Press, 2000. SenGupta DJ, Zhang B,
Kraemer B, Pochart P, Fields S, Wickens M. A
three-hybrid system to detect RNA-protein
interactions in vivo. Proc Natl Acad Sci U S A.
1996 Aug 693(16)8496-501. Weaver, RF.
Molecular Biology. McGraw Hill,2002. Websites
http//www.clontech.com/ http//www.invitrogen.co
m/ http//www.stratagene.com/