Studying DNA – Protein

Interactions
Studying DNA – Protein Interactions
• In vitro analyses of DNA – protein interactions rely on the detection of DNA –
binding protein(s) in cellular extract that can recognize specific DNA
sequence(s).
• Electrophoretic Mobility Shift Assay (EMSA), is one of these assays that
evaluate the migration patterns of DNA fragment in an electric field (either
free or bound to a protein / proteins).
• In DNA electrophoresis, DNA migrates toward the positive electrode when
subjected to a direct current (DC) electric field due to the negative charges of
the phosphate groups in the DNA molecules. Smaller sizes of DNA molecules
migrate faster than larger ones due to higher penetration ability of gel
meshwork. Protein – bound DNA molecules migrate slower than the free DNA
molecules. The retardation of DNA migration (shift), thus, indicates the
presence of DNA – protein complex. The larger this complex the greater the
retardation in gel migration of DNA (a higher shift).
• The assay requires the use of DNA – binding protein(s) (either recombinant,
in vitro expressed or extracted, in native form) with a labeled DNA fragment
(produced either by DNA cloning or by chemical synthesis; ds-oligonucleotide
probe) usually less than 300 bp long (so that it can be effectively separated
by polyacrylamide gel electrophoresis; PAGE).
EMSA; Gel Shift Assay
• In EMSA, a set of controls should be run to validate the
specificity of the DNA – protein interactions detected by
the assay.
– e.g. competition of protein binding with DNA probe using
specific and non specific non-labeled DNA (cold
competitors).
– e.g. evaluation of the formation of DNA complexes with non-
specific protein(s).
– e.g. assessing the production of higher shift (super shift)
when using multiple proteins that can bind to DNA in
different scenarios (either through single DNA – binding
protein or by accessing the DNA probe by multiple proteins.

[Link]
DNA Affinity Chromatography
• If the promoter sequence(s) of a specific gene / gene set
is/are determined, chemically-synthesized double-stranded
DNA oligonucleotide(s) is/are covalently linked to
chromatography support porous matrix (such as agarose).
The chromatography resin can then be used to selectively
bind DNA – binding proteins that recognize this sequence,
i.e. affinity purifying the DNA – binding proteins of the used
sequence.
• Although most gene regulatory proteins are present at very
low levels in the cell, enough protein can usually be
isolated by affinity chromatography to obtain a partial
amino acid sequence by mass spectrometry or protein
microsequencing analysis. Protein sequence information
can be used in database mining to identify the isolated
DNA Affinity Chromatography
A Combined EMSA / DNA Affinity
Chromatography Assay
Determining the Regulated DNA
Sequence of Unknown Gene
• If the gene(s) regulated by the DNA – binding, regulatory
protein(s) is/are unknown (unlike the case in DNA
footprinting), the purified protein can be used to select the
specific sequence, to which it/they bind(s) from a large pool of
different short DNA oligonucleotide (Oligonucleotide library).
• In this assay, a purified DNA – binding protein is allowed to
bind to a library of radio-labeled, short ds–oligonucleotides.
The dsDNA fragments that bind to the protein can be
separated from unbound fragments by EMSA, eluted off the
gel, separated from the protein(s), subjected to further rounds
of protein binding until a tightly bound dsDNA fragment(s) can
be identified by cloning and sequencing. A consensus
sequence can then be formulated, searched for in the DNA
databases to identify potential target gene(s).
Determining the Regulated DNA
Sequence of Unknown Gene
Determining the Regulated DNA
Sequence of Unknown Gene; cont.
• Another method to identify the specific DNA sequence that is recognized
by a particular DNA – binding protein, is Chromatin ImmunoPrecipitation
(ChIP assay).
• Chromatin immunoprecipitation (ChIP) is an experimental method
used to determine whether DNA-binding proteins, such as transcription
factors, associate with a specific genomic region in living cells or tissues.
• In this assay, cells are treated with formaldehyde to form covalent
crosslinks between interacting proteins and DNA, cells are then lysed,
and the crude cell extracts are sonicated to shear the DNA (to produce ~
300 bp long DNA fragments). The DNA:protein complex is
immunoprecipitated using an antibody that recognizes the protein of
interest and an antibody detection reagent bound to chromatography
resin (e.g. Protein A or Protein G agarose). The isolated complexes are
washed, then eluted and the crosslinked DNA – protein complexes are
broken apart by heating followed by protein removal by proteinase K
treatment. The remaining DNA is purified and analyzed by various ways,
including PCR, microarray analysis or direct sequencing.
Chromatin immunoprecipitation, ChIP
The ChIP Assay
• The ChIP assay helps in identifying the transcription factor
– binding sites, which may shed light on how the genes
are regulated in different cellular / organism contexts.
• In the ChIP assay,
– a crosslinking reagent such as formaldehyde is used to cross‐
link proteins to DNA in a covalent manner.
– A specific antibody is then used to immunoprecipitate the protein
of interest.
– The final product of this treatment is a pool of genomic DNA
sequences, usually ranging from 200 bp – 1 kb.
– Ideally, only sequences specifically crosslinked to the protein of
interest should be recovered after immunoprecipitation (IP), but
in practice any genomic DNA sequence binds with low affinity to
the beads used to recover the immunoprecipitated material is
also recovered. A control sample ‘‘mock’’ reaction, in which no
antibody is added during the IP, is done to check for background
DNA (non-specific interaction).
ChIP Assay (Fixation)
• Formaldehyde fixation: target tissue sample is
mildly homogenized in a buffer containing a
protease – inhibitor cocktail and formaldehyde
to a final conc. of ~ 2%.
• The tissue homogenate is incubated at room
temp to complete the crosslinking, then is spun
down at low speed and the pellet is washed
several times in homogenization buffer and
resuspended in a lysis buffer containing SDS
(0.1%) and N-lauroylsarcosine (0.5%).
• The lysed, cross-linked materials are ultra-
sonicated (to solubilize the chromatin) then spun
down to recover the chromatin extract.
ChIP Assay (Immunoprecipitation)
• Chromatin Immunoprecipitation: the
antibody/antibodies of interest is/are added to one
aliquot of the chromatin extract (~5 mg/reaction) and
incubated over night, then protein – A / protein – G
sepharose is added (to ppt the Ab complexes),
incubated, washed and eluted by heating to 65°C in
a buffer containing SDS.
• Chromatin ppt is reverse – crosslinked by heating to
65°C overnight followed by incubation with
proteinase K for several hours at 50°C.
• The mix is extracted once with phenol / chloroform
and then ethanol – precipitated and dissolved in ~20
ml water giving several nanograms of the “native”
ChIP samples.
ChIP Assay (Amplification)
• Amplification of the ChIP sample: the amount required for
hybridization with microchip is almost 1 mg, thus an
amplification step by blunt-end linker mediated PCR (LM-
PCR) is performed.
• The ChIP sample needs to be 1- phosphorylated at the 5’-
end (using polynucleotide kinase), 2- filling-in the staggered
ends (using Klenow fragment, DNA polymerase), 3- ligated
with a DNA linker [consists of two complementary
oligonucleotides annealed together: (a) a 24‐mer primer 5’‐
AGA AGC TTG AAT TCG AGC AGG ATC-3’
(phosphorylated at its 5’ end); (b) a 20‐mer primer 5’‐CTG
CTC GAA TTC AAG CTT CT-3’] (using T4 ligase).
• The ligated products are amplified using single primer – PCR
mix (primer – b, the 20-mer primer), and purified.
• The amplified IP samples are labeled by random priming (e.g.
using a Cy3 – dCTP-dNTP mix for the mock sample and Cy5
Chloramphenicol Acetyl Transferase;
the CAT Assay
• Chloramphenicol acetyltransferase (CAT), is an enzyme
that is encoded by a bacterial drug resistance gene, which
inactivates chloramphenicol by acetylating the drug at one
or both of its two hydroxyl groups.
• This gene is not found in eukaryotes, thus eukaryotic cells
produce no background CAT activity. Thus, the CAT gene
can be used for studies of mammalian gene expression.
• The regulatory sequence(s) to be studied is cloned in a
promoterless vector, which would be transfected into
mammalian tissue cultured cells to study the interaction of
the cloned regulatory sequence(s) with either endogenous
or exogenous proteins that are expressed from eukaryotic
expression vectors co-transfected with the CAT plasmid.
Chloramphenicol Acetyl Transferase;
the CAT Assay; cont.
• The interaction of transcription factors with the cloned DNA sequence(s)
would result in varied activation of the transcription of the CAT gene,
thus producing variable activities of the CAT enzyme.
• CAT activity is measured by assessing the ability of the expressed
enzyme to add acetyl group(s) to radio-actively (14C or 3H) – labeled
chloramphenicol.
• CAT reaction products can be measured either by liquid scintillation
counter (LSC) or by separating the assay products onto thin-layer
chromatography (TLC).
• In LSC, 14C– labeled chloramphenicol is incubated with cellular extracts
that may contain the CAT enzyme and with n-Butyryl Coenzyme A (n-
Butyryl CoA). CAT transfers the n-butyryl moiety to chloramphenicol.
Reaction products are extracted with xylene, where the n-butyryl
chloramphenicol partitions mainly into the xylene phase, while
unmodified chloramphenicol remains predominantly in the aqueous
phase. The organic phase is then mixed with scintillation cocktail and
different reactions are counted, which would indicate the CAT activity of
Chloramphenicol Acetyl Transferase;
the CAT Assay; cont.
• In TLC, samples are processed as described before for LSC
but extracted with ethyl acetate, at which chloramphenicol and
its buteryl derivatives are soluble. Samples are then loaded
onto silica gel TLC and chromatographic separation proceeds
(upward) in chloroform / methanol solvent system (97:3).
• Developed TLCs are covered with plastic wrap and exposed
to X-ray films (autoradiography) or processed for
phosphorimaging.
• Chloramphenicol can be modified at one or two positions
giving mono – or di – butyrylated products, which migrate
faster than the unmodified chloramphenicol on the TLC plates
in this solvent system.
• For quantitative analysis, spots on TL plates (guided by
autoradiographs) can be scraped off or cut from the plate and
counted in a scintillation counter.
Chloramphenicol Acetyl Transferase;
the CAT Assay; cont.
Studying Protein – Protein
Interactions
Studying Protein – Proteins Interactions
• Most of the work of living organisms is performed by
proteins. Proteins do their work by acting on other
macromolecules such as nucleic acids, carbohydrates,
lipids, and, especially, other proteins.
• Inside the cell, protein-protein interactions are important
in enzymatic actions upon protein substrates and in the
assembly of complexes that control signal transduction,
cell division, DNA replication, and transcription initiation
and termination.
• Outside the cell, protein interactions allow cells to talk
with each other as the case with ligands that are
expressed on the cell surface and often bind protein
receptors expressed on adjacent cells, or when
secreted protein ligands bind their receptors on distant
Studying Protein – Proteins Interactions, cont.
• Protein – protein interactions may be transient (i.e. occurs
temporarily, then the complex dissociates, e.g. enzyme –
protein substrate interaction), or stable, where proteins
participate in the formation of biological complexes, in
which proteins strongly and stably interact together and
can be isolated based on their interaction patterns.
• However, there is no particular correlation between the
stability of a protein interaction and its biological
significance, where some stably interacting proteins may
not indicate biologically significant reactions.
• Thus, it is often useful to express protein interactions in
terms of the strength, or affinity, of the interaction
between the two proteins, which is described by
equilibrium parameters, i.e. the rate at which protein
complex forms (ka) and comes apart (kd).
Equilibrium parameters
• The strength of an interaction can be given as the
equilibrium dissociation constant, Kd, or by its reciprocal,
Ka. When two proteins A and B associate, the Kd is:
Kd = AB 
AB 
Where [A], [B] and [AB] are the molar concentrations of proteins A,
B and their complex.
The higher the Kd value, the less stable the interaction is.

Examples of Kd values for

some biologically significant
interactions
GAL4 Transcription Factor
• GAL4 is a yeast transcription factor that is involved in the
regulation of galactose metabolism.
• GAL4 is a modular proteins consists of two domains, a
DNA – binding domain (DBD) and a Transcription –
activation domain (AD).
• The GAL4 upstream activation sequence (UAS), is the
DNA responsive element in the promoter region of target
gene(s), to which GAL4-DBD binds. GAL4 UAS is:
CGGN11CCG.
• In GAL4, the DBD and AD domains can be separated
from each other. When brought together (through the
interaction of different proteins fused to each of the two
domains, for example), GAL4 reassembles and regains
its transcription activation capacity.
Yeast – Two Hybrid System
• Several approaches have been developed to study protein –
protein interactions both in vivo and in vitro.
• Yeast two –hybrid is an assay, in which the interaction of two
proteins is detected by the activation of a reporter gene
expression.
• The system is designed based on the characteristic feature
of the yeast transcription factor, Gal4 to activate transcription
of responsive genes even when its DBD and AD domains of
are separated and brought together by interacting proteins
fused to each of them.
• The GAL4-DBD recognizes its responsive promoter element
(called upstream activation sequence; UAS) that can be
added upstream of a reporter gene.
• When a recombinant protein, fused with GAL4-AD, interacts
with a protein partner, fused with GAL4-DBD, this leads to
bringing the GAL4-AD next to the yeast RNA polymerase,
Yeast Two – Hybrid, the Bait
• The bait is the GAL4-DBD fused to the protein of interest.
• The major requirements for the bait protein are that it should
not be actively excluded from the yeast nucleus, and it should
not possess an intrinsic ability to strongly activate transcription.
• The sequence encoding the bait protein is cloned downstream
of, and in-frame with the GAL4-DBD encoding sequence in a
plasmid vector, e.g. pBDGAL4.
• The expression of the GAL4 – DBD and the cloned sequence
is driven by the constitutively active yeast promoter, Alcohol
dehydrogenase 1; PADH1, to assure high level of protein
expression.
• In addition to selection marker for bacteria (usually an
antibiotic resistance gene), DBD plasmid also contains a yeast
selection marker, e.g. TRP1 gene that encodes an enzyme
involved in tryptophan metabolism, phosphoribosyl-
anthranilate isomerase. TRP1 complements tryptophan (Trp)
Yeast Two – Hybrid, the Prey
• The prey, either a single protein or a cDNA library, is
the same as that used for yeast one – hybrid system.
• The sequence encoding the protein of interest (or a
uni-directional cDNA library) is cloned downstream of
the GAL4 – AD – encoding sequence in a plasmid
vector, e.g. pACT or pGAD.
• The expression of the GAL4 – AD and the cloned
sequence(s) is/are driven by a constitutively active
yeast promoter, e.g. Alcohol dehydrogenase 1; PADH1,
to assure high level of protein expression.
• The vector contains a selection marker, an auxotrophic
neutritional marker such as LEU2 gene, which encodes
b-isopropylmalate dehydrogenase and complements
leucine (Leu) auxotrophy of a leu2 mutant yeast host
Yeast Two – Hybrid, the Reporter
• Reporter gene is a gene that is either integrated
into the genome of the yeast host strain (such as
b- galactosidase reporter gene; LacZ), OR;
• Its promoter region was modified and replaced
by UASGAL4 (e.g. imidazole-glycerolphosphate
(IGP)-dehydratase; HIS3).
• The expression of the reporter gene is driven by
GAL4 upon its binding to its responsive UAS
(UASGAL4) located upstream of the reporter and
after the reconstitution of the transcription factor
when the AD-fusion interacts with the DBD
fusion, thus, providing a measure to determine
positive interaction.
Yeast Two – Hybrid, the Host
• The genome of the yeast host strains are modified in a way that
allows the selection of transformed yeast cells and detection of
protein interactions.
• For example, LEU2 and TRP1 genes (used for selection of
positive yeast colonies that are transformed with AD and DBD
plasmids, respectively) are mutated
• The promoter for HIS3 gene (used for the detection of protein
interactions) is modified by inserting UAS GAL4 upstream of the
gene.
• GAL4 and GAL80 are also mutated (inactivated) to prevent
interference from endogenous yeast genes / proteins.
• In certain host strains, the HIS3 reporter may exhibit leaky
expression in absence of induction with GAL4 due to the
promoter composition. In such cases, 3-amino-1,2,4 triazole (3-
AT), which is a metabolic inhibitor for the IGP-dehydratase
enzyme should be added to neutralize the leaky expression of
Yeast Two – Hybrid, the principle
Yeast Two – Hybrid, the System
Yeast Two – Hybrid, the Vectors
Protein – Protein Interactions;
In vitro Assays
• Several assays were designed to allow the assessment of
protein – protein interactions, (whether to validate an already
known interaction or to identify new one).
• Pulldown assays use standard affinity purification methods to
purify interacting proteins then the components of the protein
complex can be identified using a suitable detection system.
• GST pulldown assays use a GST-fusion protein bound to
glutathione chromatographic resin as a bait to affinity purify
any proteins that interact with the bait protein (thus act as
preys) from a pool of proteins in solution.
• GST fusion protein can be produced in vitro from expression
vectors such as pGEX and pET42.
• Similarly, poly-histidine fusion proteins can be used to
identify interacting proteins and they can be purified over a
Nickel chromatographic resin, which binds poly-his tag.
GST Expression Vectors
GST Pulldown Assay
• The GST fusion protein is first purified on
glutathione-agarose beads.
• The bead-bound fusion protein is then used
as “bait” to evaluate the binding of a known or
suspected “test” protein which may be either
purified or labeled by in vitro translation.
• Beads with bound protein are washed, and
the retained test protein is determined
– by elution with glutathione OR
– by separation onto SDS-PAGE and detection by a
suitable method.
GST Pulldown Assay
Pulldown Assay
Protein – Protein Interactions;
In vitro Assays; cont.
• Co-Immunoprecipitation (Co-IP) is another technique to
examine protein – protein interactions.
• In this assay, a protein of interest, to which an antibody reagent
is available, is used as a bait to isolate interacting proteins (the
prey).
N.B. A tag – specific antibody can be used in this assay with its fusion
protein as a bait.
• The protein complex is isolated by the addition of the bait –
specific antibody.
• The antigen – antibody complex can be purified by the addition
of chromatographic resin of Protein A (a surface protein from
Staphylococcus aureus that binds the Fc region of
immunoglobulin Gs; IgGs, from many species) or Protein G
(an immunoglobulin – binding protein expressed on the surface
of group C and G streptococcal bacteria).
• The resin is washed and separated onto SDS-PAGE and
Co-Immunoprecipitation Assay