Proteinprotein interactions in cells:

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Last updated Nov. 7, 2006
Protein-protein interactions in
cells Co-immunoprecipitation (co-IP) from
extracts 2-hybrid formation FRET (Fluorescence
resonance energy transfer) Complementation
readout
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Yeast Two Hybrid Assay
Activates transcription locally
Candidate binding protein
Binds DNA
Bait- test protein
Reporter
U. Arizona
DB DNA binding domain AD activation domain
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Protein-protein interaction within a yeast cell.
Alternative detection methods
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Single bait vector
Library of cDNAs in target vector
Already in yeast with reporter genes
Transform into yeast
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(No Transcript)
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Replica plate
HIS3 for growth
lacZ for color
Limitation often false positives.
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3-HYBRID select for proteins domains that bind
a particular RNA sequence
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REVERSE TWO HYBRID
Selection against reporter gene allows
identification of altered proteins (mutants) that
no longer interact Or third proteins or
chemicals that disrupt a protein-protein
interaction.
blue
white
U. Arizona
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Yeast one-hybrid Insert a DNA sequence
upstream of the selectable or reporter Transform
with candidate DNA-binding protein fused to an
activator domain.
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FRET Fluorescence resonance energy transfer
(Excite with this)
Emission at 530 nm
(Direct, no photons)
(measure this)
Excitation At 530 nm
The closer the fluorophores are to each other,
the greater the FRET Distances up to 100 A can
be detected Changes down to 2 A can be
measured Intramolecular distances and their
changes can be measured with FRET Usually
measured in a fluorometer FRET can be seen in a
fluorescent microscope
Emission at 570 nm
YFP Yellow fluorescent protein CFP Cyan
fluorescent protein
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The role of Drf3 in Cdc42-regulated actin
remodelling
Blue in, Green out Green in,
Red out Blue in, red out FRET
Diaphanous-related formins (Drfs) act as Rho
small GTPase effectors during factor-induced
cytoskeletal remodelling.
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SW Michnick web site http//michnick.bcm.umontrea
l.ca/research/images/pca_general_en.gif
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Seminar Dec. 6, 2006 at 12 noon 700 Fairchild
From the assigned reading
FKBP, FK506 binding protein FRAP, FKBPrapamycin
binding proteinFRB, FKBPrapamycin binding
domain of FRAP
DHFdihydrofolate FH2 THFtetrahydrofolate
FH4 fMTXfluorescent methotrexate
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General model for transcriptional regulation in
higher eukaryotes
TF transcription factor TBP TATA binding
protein TAF TBP associated protein BRE TFIIB
response element
INR transcription initiator element DPE
downstream promoter element
The transcription complex either recruits RNA
PolII or activates a bound RNA PolII
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(No Transcript)
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Many enhancer elements often lie upstream of
promoters,allowing for many combinations of TF
binding
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Put a DNA regulatory region upstream of a
reporter gene to analyze its elements
Space for res. enz. to bind
PCR
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Popular reporters to study promoter/enhancers

• Beta-galactosidase (B-gal) detection
• Chloramphenicol acetyl transferees (CAT)
  detection, sensitive
• Luciferase (firefly, Renilla jellyfish)
  detection, easy dual
• Green fluorescent protein (GFP, BFP, YFP))
  cytological
• Neomycin phosphotransferase (neo)selectable
  (G418R)
• Dihydrofolate reductase (DHFR) selectable,
  amplifiable

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Testing for a cell-specific promoter
chloramphenicol acetyl transferase (CAT) reporter
assay
CAT cDNA is from a prokaryotic source. CAT is
not found in mammalian cells. Therefore low
backgrounds
diacetylated
B
A
Thin layer chromatography (TLC)
monoacetylated
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Reporter enzyme substrates for different purposes
Substrates for beta-galactosidase, for example

• ONPG (ortho-nitrophenyl-beta-galactoside)
  spectrophotometric measurement (420 nm blue
  color simplest)
• X-gal (5-Bromo-4-chloro-3-indolyl-ß-D-galactoside)
  blue precipitate - for cytology or colony
  detection
• Umbelliferylgalactoside (-gt umbelliferone,
  fluorescent, reading in a fluorimeter allows more
  sensitive quantification than spectrophotometry)
• Galacton-STAR (-gt chemiluminescent product
  emission of light, so lower background than
  fluorescence)
• Lactose (glucose-beta-galactose disaccharide)
  allows growth if hydrolyzed growth phenotype.
  For microbial cells usually.

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Mapping transcriptional elements upstream of a
promoter
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FootprintingTo detect sites on DNA to which
protein are bound
DNA DNA-binding protein
Naked DNA
Population of molecules
Population of molecules
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Note uneven cleavage of naked DNA by DNase
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Got this far
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Methylation Interference
Isolate DNA bound to protein (e.g., on
nitrocellulose filter binds protein but not DNA)
Ross Hardison Lab, Penn State
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Methylation Interference
Partially modify bases (G, A) by methylation of
pure labeled DNA fragment (E.G., WITH DIMETHYL
SULFATE). DNA will be hit randomly and partially.
1.
Bind DNA to protein and separate DNA that is
bound to protein (so one is looking only at the
DNA molecules that still can bind the
proteindespite being methylated)
2.
3. DNA molecules that cannot bind to protein
because they happen to have been
methylated at the protein target site on DNA will
NOT be present
Naked DNA control
4. Cleave DNA at methylated sites See almost
all sites hit ? band, but no bands for
positions where methylation prevented protein
binding (escaped being isolated via the protein
binding criterion) - looks like a footprint.
DNA bound to protein can be isolated by binding
to nitrocellulose Proteins and
DNA-proteincomplexes bind, naked DNA passes
through
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Protein-DNA binding EMSA or gel shift
(EMSA electrophoretic mobility shift assay)
1 2 3 4 5
(supershift)
(shift)
DNA element
(Even though the hexagon looks like a protein
here)
U. Arizona
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Gel shifts
Protein DNA complexes migrate more slowly than
naked DNA
(competed only by specific probe)
(two molecules of protein bound)
(competed by NON-specific probe)
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Synthetic, range usually 6 to 40-mers
SELEX
(T7 RNA Pol from an embedded T7Pol promoter
Systematic evolution of ligands by exponential
enrichment
(huge number)
(usually a protein)

by PCR
(re-iterate 3-10 times)
Binding to Protein, e.g.
Separate using nitrocellulose binding, gel
electrophoresis, etc.
sequences ? consensus
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Binding site for a puf protein, implicated in
mRNA degradation
PUM2, a novel murine puf protein, and its
consensus RNA-binding siteWhite EK,
Moore-Jarrett T, Ruley HE. RNA. 2001
Dec7(12)1855-66.
Consensus
Description