CG920 Genomics
Lesson 6
Protein Interactions in Gene Regulations
Jan Hejátko
Functional Genomics and Proteomics of Plants,
Mendel Centre for Plant Genomics and Proteomics,
Central European Institute of Technology (CEITEC), Masaryk University, Brno
hejatko@sci.muni.cz, www.ceitec.muni.cz
 Literature sources for Chapter 06:
 Wilt, F.H., and Hake, S. (2004). Principles of Developmental Biology. (New York ;
London: W. W. Norton).
 Ainger, K., Avossa, D., Morgan, F., Hill, S.J., Barry, C., Barbarese, E., and Carson, J.H.
(1993). Transport and localization of exogenous myelin basic protein mRNA microinjected
into oligodendrocytes. J Cell Biol 123, 431-441.
 Alberts, B. (1998). The cell as a collection of protein machines: preparing the next
generation of molecular biologists. Cell 92, 291-294.
 Grefen, C., Stadele, K., Ruzicka, K., Obrdlik, P., Harter, K., and Horak, J. (2008).
Subcellular localization and in vivo interactions of the Arabidopsis thaliana ethylene
receptor family members. Molecular Plant 1, 308-320.
 Hu, C.D., and Kerppola, T.K. (2003). Simultaneous visualization of multiple protein
interactions in living cells using multicolor fluorescence complementation analysis. Nat.
Biotechnol. 21, 539-545.
 Shahbabian, K., and Chartrand, P. (2012). Control of cytoplasmic mRNA localization.
Cellular and molecular life sciences : CMLS 69, 535-552.
 Van Leene, J., Witters, E., Inze, D., and De Jaeger, G. (2008). Boosting tandem affinity
purification of plant protein complexes. Trends Plant Sci 13, 517-520.
 Walter, M., Chaban, C., Schutze, K., Batistic, O., Weckermann, K., Nake, C., Blazevic, D.,
Grefen, C., Schumacher, K., Oecking, C., Harter, K., and Kudla, J. (2004). Visualization of
protein interactions in living plant cells using bimolecular fluorescence complementation.
Plant J 40, 428-438.
Literature
2
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Outline
3
 Functional importance of specific protein
interactions
 Most of the proteins in the cell exist in the
form of complexes which may further
interact with each other
 Proteasome
 protein complex responsible for the
degradation of obsolete proteins in
the cell
The importance of
protein interactions
4
Once obsolete proteins are tagged with at least four ubiquitin molecules, they are destroyed by
proteasomes. Proteasomes are voracious protein shredders, but the destructive machinery is
carefully protected so that it can't attack all of the normal proteins in the cell. The proteasome,
shown here from PDB entry 1fnt, is shaped like a cylinder, with its active sites sheltered inside the
tube. The caps on the ends regulate entry into the destructive chamber, where the protein is
chopped into pieces 3 to 23 amino acids long.
Most of the non-lysosomal proteolysis that occurs in eukaryotic cells is performed by a
nonspecific and abundant barrel-shaped complex called the 20S proteasome. Substrates access
the active sites, which are sequestered in an internal chamber, by traversing a narrow opening
(alpha-annulus) that is blocked in the unliganded 20S proteasome by amino-terminal sequences
of alpha-subunits. Peptide products probably exit the 20S proteasome through the same opening.
11S regulators (also called PA26 (ref. 4), PA28 (ref. 5) and REG) are heptamers that stimulate
20S proteasome peptidase activity in vitro and may facilitate product release in vivo. Here we
report the co-crystal structure of yeast 20S proteasome with the 11S regulator from Trypanosoma
brucei (PA26). PA26 carboxy-terminal tails provide binding affinity by inserting into pockets on the
20S proteasome, and PA26 activation loops induce conformational changes in alpha-subunits that
open the gate separating the proteasome interior from the intracellular environment. The
reduction in processivity expected for an open conformation of the exit gate may explain the role
of 11S regulators in the production of ligands for major histocompatibility complex class I
molecules. (PDB, Whitby et al., (2000) Nature 408: 115-120,
http://www.rcsb.org/pdb/explore/explore.do?structureId=1fnt).
5
 Functional importance of specific protein interactions
 Chromatin structure
The importance of
protein interactions
6
Regulation by histone
acetyl transferases or
histone deacteylases
7
Regulation of the chromatin structure represents one of the very basal gene
expression regulatory levels. Chromatin is a substrate for DNA-dependent RNA
polymerases that transcript the DNA encoded information into the “words and
sentences” of RNA.
Regulation of chromatin structure and its accessibility to DNA-dependent RNA
polymerases depends on many factors, one of the most important is the regulation
of chromatin binding to nucleosomes and chromatin methylation.
Regulation of chromatin interaction with histones, the positively charged proteins
forming the core of nucleosomes, is performed via modification of acetylation status
of the N-terminal portion of histones, especially histones H3 and H4. This occurs
via action of histone acetyl transferases or histone deacteylases.
8
CpG or CpNpG
CpNpNp
CpG
DNA methylation in animals vs. in plants
methylation status methylation status
Cell-specific methylation allows maintain of
tissue-specific gene expression profiles
Mechanism of transcriptional regulation by
DNA methylation mostly unknownImprinting and “cell memory”
9
Modification of the chromatin methylation is performed via DNA methyltransferases.
Interestingly, there is difference in the methylation in animals and in plants.
In animals, the methylation takes place mostly on the cytosine that occurs next to guanosine (the
sequence is denoted as CpG). In mammals, 60-90% of all CpGs are methylated.
In plants, cytosines are methylated both symmetrically (CpG or CpNpG) and asymmetrically
(CpNpNp), where N is any nucleotide.
Methylation status is usually “reset” in the zygote and is reconstituted during development again.
E.g. the methylation is very low in the mouse embryo at the blastula stage, however, DNA derived
from later stages when organogenesis is initiated is substantially more modified by methylation.
DNA methylation also stably alters the gene expression pattern in cells such that cells can
"remember where they have been"; in other words, cells programmed to be pancreatic islets
during embryonic development remain pancreatic islets throughout the life of the organism
without continuing signals telling them that they need to remain islets.
DNA methylation is involved in the genomic imprinting, i.e. the genes originating from both
parents are often diversely methylated, which results into differential expression of parental
genomes (for the importance of the imprinting in the parental conflict and epigenetics, see the
lecture “Bi0580 Developmental genetics” by prof. Vyskot).
Up to know it is not clear how methylation regulates transcription. Possibly, methylation status
affects chromatin configuration or binding general repressor factors.
10
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
Importance of PI
11
Formation of transcription initiation complex
Regulation of transcription
occurs via specific interaction
of both general and tissue
specific transcription factors
(TFs) with promoter and/or
enhancer sequences.
The scheme above shows
simplified subsequent
formation of the complex of
TFs involved in the
regulation of transcription.
Interaction of general TFIID
with the TATA box induces
distortion of the DNA
structure (see the next slide).
12
Induction of structural
changes upon interaction of
TFIID with DNA. This may
be important for the
assembly of other TFs
involved in the formation of
transcription initiation
complex.
This change of confirmation
provides a kind of
“signature” that is
recognized by other
proteins and NA
polymerase to recognize
the proper binding site.
However, there are also
TATA box-less promoter,
where probably other types
of “signatures” occur.
13
Positive TFs
Negative TFs
Formation of transcription initiation complex
14
The scheme showing the formation of the transcription initiation complex and the
interaction of both positive (open symbols) and negative (solid symbols) factors.
These proteins bind to the regulatory sequences that might be hundreds or even
thousands of base pairs away from the promoter. These protein interact with each
other and with the RNA polymerase, integrating thus many signals into a “yes” or
“no” response of the basal promoter, i.e. the region adjacent to the TATA box and
recognized by the RNA polymerase.
The individual positive or negative factors are complex and their activity might be
regulated by their phosphorylation status or via their interaction with other proteins
(i.e. momomeric or dimeric) etc..
15
Mechanism of transcriptional
regulation by TAFs
Signal recognition
Dimerization
DNA binding
and
transcription
activation every 7th aa
16
There is a whole family of transcription activating factors (TAFs) that interact with
signalling molecules, e.g. steroid hormones, thyroid hormones or retinoic acid and in
a response to the signal transfer to the nucleus where they regulate transcription.
One of the type of TAF are leucine zipper or bZIP type TAFs. These TAFs are
dimeric, with leucine-rich hydrophobic face formed by the Leu that occurs every 7th
aa.
That allows the factor to take the proper configuration, which provides the dimer with
the ability to bind DNA via charged aa.
17
“Microprocessor-like” acting promoters
ProENDO16:REPORTER (sea urchin)
Deletion
mutagenesis Positive, interaction with
TAFs
Upregulation in the
presence of A and B
Developmental specificity
Combinatorial control
Midgut
Primary or skeletogenic
mesenchyme cells
Vegetal plate
18
An example of the “microprocessor”-like acting promoter is a promoter of the endo16 gene from
the sea urchin.
There have been identified several gene regulatory modules in the endo16 gene that have
positive or negative regulatory role. These modules were identified via formation of deletion
mutants of the transcriptional fusions with reporter gene.
The analysis has revealed that the module A has a positive function and must interact with its
cognate TAFs for transcription to occur.
Module G enhances the expression when the A and B are active.
C, D, E and F are responsible for the specificity of the expression of endo16 during sea urchin
development.
Each of the modules has several protein interaction sites, some of them general, other unique.
Site for the protein SpGCF1 is present in many modules and is probably responsible for looping
of chromatin, allowing thus bringing of distal regulatory modules close to the basal promoter.
This type of regulation, i.e. based on the different activities of diverse regulatory sequences is
sometimes called combinatorial and is common for development of many living creatures.
In the combinatorial type of regulations, some modules may act synergistically, some of them
antagonistically, some may have both positive and negative roles (e.g. the module B, see the
figure). This variability allows very precise and responsive regulations towards changing
environmental conditions.
19
“Microprocessor-like” acting promoters
Regulation of β-globin type of
hemoglobin chains expression
Locus control region
Development-dependent
activation by LCR
•Acetylation of H3?
•Involvement of other
genes?
Cca 50 kbp
20
An example of the combinatorial gene regulation is the regulation of β-globin type of
hemoglobin chains of humans.
As discussed in the Lesson 5 (course Bi8940 Developmental biology), the type of hemoglobin
produced by the fetus changes during development. The hemoglobin present in the liverproduced
hemoglobin is composed of two α- and two β-type chains. The β-type hemoglobin
chains are of several developmental types, produced by ε, γ1, γ2 and β (in this order). In
addition, there is minor adult type of β-type hemoglobin, called δ globin.
The genes for the β-type chains are aligned on the chromosome in the order, in which they
are expressed during development (see the figure).
For the expression of individual cell types is distinctive an upstream regulatory sequence
called locus control region (LCR). LCR is located about 50 kbp away from the most proximal ε
gene.
21
The LCR structure is different in erytrocyte precursor cells in comparison to other cells that
could be demonstrated by the changes in the sensitivity to low concentrations of DNase,
suggesting low amount of nucleosomes bound.
For the expression of the particular genes, the interaction of their regulatory sequences with
LCRs is necessary. Because of LCR can interact only with one regulatory sequence at a time,
only one type of genes for the particular β-type chain is activated (the first interaction of LCR
with ε gene, which is later in development replaced by the other one, is shown by the doubleheaded
arrow).
The underlying molecular mechanisms of the specific pattern of the LCR movement from the
most proximal towards the most distal gene cannot be satisfactory explained.
Probably, acetylation of H3 histones might play a role and possibly, other genes outside of the βtype
chain family are involved in the regulation of LCR activity. That seems to be confirmed by
the identification of other human genes with similar structure, suggesting common regulatory
mechanisms via LCRs.
22
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
Importance of PI
23
BICOID mRNA NANOS mRNA
 Importance of mRNA localization
 Spatiotemporal localization of gene
product (protein)
 Asymmetric cell division during
development
 Polarization of embryo
mRNA localization
Shahbabian and Chartrand, 2012
ASH1 mRNA
24
 Role of mRNA localization
 Downregulation of expression
of potentially toxic proteins
 Localization of
expression of MBP into
myelination regions of
nerve cells
mRNA localization
Ainger et al., 1993
MBP mRNA
25
Myelin basic protein (MBP) is a protein believed to be important in the process of myelination
of nerves in the nervous system.
The images show localization of mRNA for MBP. Digoxigenin-labeled MBP RNA was
microinjected into mouse oligodendrocytes growing in primary culture. The injected RNA
appeared as small granules which were present throughout the cytoplasm and processes,
and was also found dispersed in the peripheral membranes of the cell.
To analyze the three dimensional distribution of microinjected labeled MBP mRNA throughout
the cell, consecutive optical sections through a single oligodendrocyte were collected,
reconstructed, and visualized using volume rendering (Fig. A) or isosurface rendering (Fig. B)
techniques. An oligodendrocyte microinjected with MBP mRNA, visualized by volume
rendering is shown in Fig. A. RNA granules were observed throughout the perikaryon and in
some, but not all, processes. The granules in the perikaryon and in the processes appeared
to be equivalent in size. In some regions the granules in the processes were aligned in tracks.
Although not apparent from this image, the nucleus was devoid of granules.
26
Shahbabian and Chartrand, 2012
 Diffusion-entrapment mechanism of mRNA
 During the early stages of Xenopus
oogenesis, Xcat-2 mRNA is restricted to
a specific structure in the cytoplasm
called the mitochondrial cloud (MC,
Balbiani body)
 MC movement is partly dependent on
the depolymerization of microtubuls (socalled
„molecular motor“)
 Entrapment on the vegetal pole occurrs
because of interaction of MC and ER
mRNA localization
Mechanisms
27
Another well studied example of the diffusion-entrapment mechanism is the Xenopus
Xcat-2 mRNA, which encodes a Nos related zinc-finger RNA-binding protein.
During the early stages of Xenopus oogenesis, Xcat-2 mRNA is restricted to a specific
structure in the cytoplasm called the mitochondrial cloud (MC). The mitochondrial cloud,
also called Balbiani body, consists mostly of mitochondria and small vesicles, and is the
source of germinal granule material [32]. The movement of the MC in the cytoplasm
results in the localization of the Xcat-2 mRNA at the vegetal cortex (Shahbabian and
Chartrand, 2012).
28
 Localized mRNA degradation
 During embryogenesis in
Drosophila m. Hsp83 mRNA is
localized at the posterior pole of
embryo, similarly to NANOS
mRNA
 Hsp83 mRNA is localized in the
whole embryo, however, it is
destabilized by cis elements both
in 3’UTR (HDE) and in coding
region (HIE).
mRNA localization
Mechanisms
 HIE elements are recognized by SMAUG protein, which
mediates binding of degradation complex CCR4/POP2/NOT
 In the posterior pole the Hsp83 mRNA is protected from the
effects of SMAUG by the so-called HPE element in 3’UTR;
mechanism of this protection has been still unknown
Shahbabian and Chartrand, 2012
29
Localized stabilization of a transcript is another mechanism by which an mRNA can be
subcellularly targeted. In this case, an mRNA is rapidly degraded in most parts of the cell, but it
is protected from degradation at a specific location. The hsp83 mRNA, which encodes a heat
shock protein in Drosophila, is a well-characterized example of this kind of localization (Fig.
2b). This transcript is localized at the posterior pole of the early Drosophila embryo by the
selective stabilization of the mRNA at the posterior pole and degradation of the transcript
elsewhere in the cytoplasm.
The level of hsp83 mRNA, which is a maternally encoded transcript, decreases more rapidly in
embryos than in unfertilized eggs, which suggests that two separate mechanisms control the
stability of this transcript [38]. These two independent pathways, which are called ‘‘maternal’’
and ‘‘zygotic’’ pathways, use maternally and embryonic encoded proteins, respectively, to
degrade the hsp83 transcript [38]. By analyzing the 3’UTR of hsp83 mRNA, a region from
nucleotides 253–349 was identified as the Hsp83 degradation element (HDE), which directs
the destabilization of this mRNA in unfertilized eggs. However, this region has no effect in the
zygotic degradation pathway, and transcripts without the HDE domain are subject to
degradation by the embryonic degradation machinery [38].
30
The hsp83 ORF has also been shown to affect the stability of the transcript. A region at the 3’
end of the ORF, which comprises 615 nucleotides, has been found to be responsible for this
destabilization, and was consequently called Hsp38 instability element (HIE) [39]. This region,
which has the major effect in the destabilization of the transcript, functions together with the
HDE for complete degradation. The HIE domain contains six stem-loop structures that are
recognized by the maternally encoded RNA-binding protein Smaug [39, 40]. It was shown that
in Smaug mutants, degradation and thus localization of hsp83 mRNA are impaired. Smaug
recruits the CCR4/POP2/NOT deadenylase complex, triggering deadenylation and thus
degradation of the hsp83 transcript [40]. Although Smaug is present throughout the pole plasm,
the hsp83 mRNA is protected from Smaug action at the posterior pole. This protection is
related to a 57 nt region in the 3’UTR (nucleotides 351–407) downstream of HDE, which is
called HPE (Hsp83 protection element). HPE is sufficient to confer stability to an unstable
transcript at the pole plasm [40]. The mechanism by which this domain functions is not clear,
and may include interaction of trans-acting factors that block the availability of the transcript to
Smaug (Shahbabian and Chartrand, 2012).
31
Shahbabian and Chartrand, 2012
 Active transport of mRNA
 ASH1 is represor of the HO
endonuclease in S. cereviseae;
inhibition of HO results in inhibition
of mating-type switching in daughter
cells
 ASH1 mRNA is actively transported
by „molecular motors“ associated
with actin
mRNA localization
Mechanisms
 ASH1 mRNA contains 4 cis
elements (3 in the coding
sequence and 1 in the
3’UTR), which are recognized
by RNA-binding protein SHE2
 SHE2 interacts with SHE3, an
adaptor protein, which links
SHE2 to the molecular motor
MYO4, which then binds to
actin and allows transport of
ASH1 mRNA into the
daughter cell
Shahbabian and Chartrand, 2012
ASH1 mRNA
32
Localization of ASH1 mRNA is essential for the asymmetric distribution of Ash1, which acts as
a transcriptional repressor of the HO endonuclease and results in inhibition of mating-type
switching in daughter cells [88, 89]. The ASH1 mRNA contains four localization elements, three
in the coding sequence (E1, E2A, and E2B) and one overlapping the end of the coding
sequence and the 3’UTR (E3) [25, 90]. While the presence of these four elements leads to an
optimal localization, deletion analysis revealed that each element is sufficient for localization of
a reporter mRNA to the bud.
When each of these elements was inserted in multiple copies in the 30UTR, the new
constructs showed nearly normal localization. However, for these mRNAs, the asymmetric
distribution of Ash1 was impaired, suggesting that the position of these elements is important
for Ash1 sorting but not for ASH1 mRNA localization [91]. Although the primary sequences of
the four ASH1 localization elements are different, they all fold into a stem-loop structure that
contains a few conserved nucleotides [92, 93]. All four elements interact with the same RNA
binding protein called She2, which is involved in the localization of bud-localized mRNAs in S.
cerevisiae.
33
She2 forms a tetramer under physiological conditions, and mutations that disrupt this
tetrameric state abolish its RNA-binding capacity and impair She2-dependent localization to
the bud tip [94]. She2 interacts directly with the C-terminal domain of She3, an adaptor
protein that links the She2–mRNA complex to the molecular motor Myo4 (Fig. 2c) [55, 95,
96]. Recent evidence also suggests that She3, besides its role in connecting the She2–RNA
complex to Myo4, is itself able to bind RNA and acts synergistically with She2 to increase
the affinity and specificity of RNA binding [97].
Recent studies on Myo4 helped to explain why multiple localization elements are required
for proper ASH1 mRNA localization. Myo4 is a class V myosin whose main function is the
transport of mRNAs to the bud tip using actin filaments [98–100]. Myo4, unlike other type V
myosins, is a nonprocessive monomer in vivo, but it becomes processive when present in
the form of oligomers [101, 102]. Purification of the localization complex associated with a
single localization element revealed that multiple copies of Myo4 are associated with this
RNA [103]. Moreover, increasing the number of Myo4 attached to the ASH1 mRNA
increased the efficiency of localization of this transcript. These results suggest that each
localization element interacts with higher order protein complexes in which a She2 tetramer
may recruit multiple copies of Myo4, thus ensuring a continuous and processive movement
of the mRNP complex into the bud. Moreover, it is possible that a She2 tetramer binds
simultaneously to the localization elements of a single transcript or, alternatively, to those of
different mRNAs. This would bring multiple mRNAs together within a single complex in
which several Myo4 molecules modulate their transport to the bud tip (Shahbabian and
Chartrand, 2012).
34
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
Importance of PI
35
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
Importance of PI
36
MP/ARF5
BDL/IAA12
Capron et al., Arabidopsis Book (2009)
Auxin signalling and its role in the embryo patterning
37
Scheme of the auxin signaling pathway as an example of the role of protein
stabilization leading to regulation of gene expression.
Under low intracellulr auxin concentrations, the transcription activators of auxinregulated
genes, which are called auxin responsive factors (ARFs), are in a complex
with negative regulators of transcription, so called AUX/|IAA proteins. In the complex,
Arfs can not activate transcription.
After auxin is imported into the cell, it binds to the TIR1 protein, that allows interaction
with AUX/IAA-ARF complex and targets AUX/IAA protein for the degradation via
proteosome.
That allows ARFs to enter nucleus and activate trancription of auxin-induced genes.
38
 Functional importance of specific protein interactions
 Chromatin structure
 Regulation of transcription
 mRNA localization
 hnRNA splicing
 Protein stability
 Signal transduction
Importance of PI
39
 PI and signal transduction
 through G protein and
phospholipase C
 Signalling cascades using cAMP
PI and signal transduction
40
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
Outline
41
PI in vivo
Co-immunoprecipitation
 Based on the isolation of protein complexes
using antibodies recognizing one of the
interacting proteins
 The principle of co-immunoprecipitation is
used in a method, which confirms
interactions of proteins, which is already
assumed by the pull-down assay
CKI1
MYC
CKI1
HA
αMYC
αHA
CKI1
MYC
AHK3
HA
αMYC
42
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
Outline
43
PI in vivo
Tandem affinity purification (TAP-tag)
 Isolation of protein complexes using recombinant
proteins fused with two different binding domains
 Isolated protein complexes are divided using 1D
ELFO and then identified by MS
 calmodulin-binding protein (CBP)
 IgG binding domains of protein A (ProtA)
 TEV (tobacco etch virus) protease
recognition site
POI
 Advantage: using two independent protein
domains for affinity purification -> therefore high
specifity
44
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
Outline
45
PI in vivo
Yeast two-hybrid assay (Y2H)
 Isolation of protein complexes using recombinant
proteins, each fused to a part of Gal4 transcription
factor
 One of the proteins (bait) fused to DNAbinding
domain of Gal4 (Gal4-BD)
 The other protein (prey) fused to
activation domain of Gal4 (Gal4-AD)
 Protein interactions enable reconstitution of
binding domains with activation domain and
triggers the expression of a reporter gene
 Visual detection (blue color, LacZ)
 Auxotrophic selection (growth on
medium lacking histidine, His)
 Method used for searching interaction partners in
expression libraries of individual organisms
46
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
Outline
47
 Protein interaction is detected by
reassociation of the fluorescent protein
 Each of the potential interaction
partners is fused to one of the
subunits of the fluorescent protein,
e.g. YFP
 In case of interaction, the
fluorescence reappears
 Apart from identification of the interaction,
this method allows you to locate the
interaction in the cell
PI in vivo
Bimolecular fluorescence
complementation (BiFC)
48
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
Outline
49
PI in vivo
Membrane Recruitment Assay (MeRA)
 Method for identification of interactions of
cytoplasmic proteins with the membrane proteins
 Membrane protein is fused with a
fluorescecnt protein
 Potential interaction partner is fused
with another fluorescent protein with
different emission spectra
 In case of interaction the localization of
the cytoplasmic protein is changed – it
is colocalized on the membrane with
the membrane protein
50

PI in vivo
Membrane Recruitment Assay (MeRA)
51
GFP
GFPGFPGFP
P35S::ERS1:RFP + P35S::ΔTM-ETR2:GFP
PI in vivo
Membrane Recruitment Assay (MeRA)
52
 Functional importance of the specificic interactions of
proteins in the regulation of gene expression
 Chromatin structure
 Regulation of transcription
 mRNA localization
 mRNA stability
 Protein stability
 Signal transduction
 Methods of analysis of protein interactions in vivo
 Co-immunoprecipitation
 The tandem affinity purification (TAP-tag)
 Yeast two-hybrid assay (Y2H)
 Bimolecular fluorescence complementation (BiFC)
 Membrane Recruitment Assay (MeRA)
 Practical use of methods for in vivo studies of protein
interactions
Outline
53
D’Agostino et al., Plant Phys, 2000
Signal Transduction via MSP
NUCLEUS
PM
AHK sensor histidine kinases
• AHK2
• AHK3
• CRE1/AHK4/WOL
REGULATION OF TRANSCRIPTION
INTERACTION WITH EFFECTOR PROTEINS
HPt Proteins
• AHP1-6
Response Regulators
• ARR1-24
CK primary response genes
- Type-A ARRs expression
Recent Model of the CK Signaling via Multistep Phosphorelay (MSP)
Pathway
54
AHP1
AtHK1
AHP2
AHP3
AHP4
AHP5
AHK2
AHK3
AHK4
CKI1
CKI2
ETR1
ETR2/EIN4
Is there any specificity in plant
MSP?
□ Is there a signalling specificity of MSP in plants?
55
Specificity of CKI1 signalling
□ CKI1 interacts in vivo with only subset of AHPs
BiFC Y2H
AHP1
AHP2
AHP3
AHP4
AHP5
AHP6
CKI1 CKI1RD
Pekárová et al., Plant Journal (2011)
56
Specificity of CKI1 Signalling
□ Specificity of CKI1 interaction was confirmed in vitro
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0 50 100 150 200 250
AHP concentration [nM]
A450
AHP2 AHP3 AHP5
AHP3: Kd ~ 10,5 nM
AHP2: Kd ~ 9,17 nM
AHP5: Kd ~ 108 nM
Pekárová et al., Plant Journal (2011)
57
Structure of CKI1RD
□ X-ray crystallography revealed conserved (α/β)5 structural fold
of CKI1RD
Pekárová et al., Plant Journal (2011)
58
Dynamics of CKI1RD
□ Mg2+binding leads to remodelling of active centre of
CKI1RD
Pekárová et al., Plant Journal (2011)
59
CKI1RD structural changes are
associated with its binding specificity
□ Mg2+- and BeF3
--induced structural changes fine-tune
binding specificity of CKI1RD
Pekárová et al., Plant Journal (2011)
60
AHP1
AtHK1
AHP2
AHP3
AHP4
AHP5
AHK2
AHK3
AHK4
CKI1
CKI2
ETR1
ETR2/EIN4
Model Suggestion
□ YES, there is signalling specificity of MSP in plants.
P
P
P
P
P
P
P
61