Proteinprotein interactions in cells:

1
Protein-protein interactions in
cells Co-immunoprecipitation (co-IP) from
extracts 2-hybrid formation Complementation
readout FRET (Fluorescence resonance energy
transfer)
2
Yeast 2-hybrid test
3
Replica plate
Beta-gactosidase chromogenic substrate
HIS3 for growth
Limitation often false positives.
4
Yeast one-hybrid Insert a DNA sequence
upstream of the selectable or reporter Transform
with candidate DNA-binding proteins (e.g., cDNA
library) fused to an activator domain.
Each T one copy of a DNA target sequence
5
3-HYBRID select for proteins domains that bind
a particular RNA sequence
6
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
7
Directed Evolution of a Glycosynthase via
Chemical Complementation Hening Lin, Haiyan Tao,
and Virginia W. Cornish J. AM. CHEM. SOC. 2004,
126, 15051-15059
8
Survivors are enriched for cellulase genes that
will cleave cellulose with greater efficiency
(kcat / Km)
Yeast cell
Toxic protein gene
cellulase
Directed Evolution of Cellulases via Chemical
Complementation." P. Peralta-Yahya, B. T. Carter,
H. Lin, H. Tao. V.W. Cornish. submitted.
x
x
x
x
Library of cellulase mutant genes (one per cell)
9
F reporter protein fragment
SW Michnick web site http//michnick.bcm.umontrea
l.ca/research/images/pca_general_en.gif
10
Clonal selection and in vivo quantitation of
protein interactions with protein-fragment
complementation assays, I. Remy and S.W. Michnick
PNAS 96, 3945399, 1999
From the assigned reading
DHFR fragments
fMTX
FKBP FK506 binding protein FRAP
FKBPrapamycin binding proteinFRB
FKBPrapamycin binding domain of FRAP
DHFdihydrofolate FH2 THFtetrahydrofolate
FH4 fMTXfluorescent methotrexate
11
a phosphatase
12
Can detect 0.05 nM rapamycin ??
13
Leucine zipper protein fragments instead
of rapamycin binding proteins
Background association of FKBP and FRB without
rapamycin
(compare)
14
(No Transcript)
15
8-fold increase in fluorescence per cell
Competition with a molecule that binds only one
16
Erythropoietin-erythropoietin receptor (dimer)
interaction
EMP1 Erythropoietin mimetic peptide 1
17
FRET
http//mekentosj.com/science/fret/fret.html
18
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
19
FRET example The role of particular proteins in
regulated actin remodellingat the edge of a cell
membrane
Blue light in, Green out Green light in,
Red out Blue light in, red out FRET
Diaphanous-related formins (Drfs) act as Rho
small GTPase effectors during factor-induced
cytoskeletal remodelling.
20
General model for transcriptional regulation in
higher eukaryotes
Core transcriptional elements
TF transcription factor TBP TATA binding
protein TAF TBP associated protein BRE TFIIB
response element
INR transcription initiator element DPE
downstream promoter element
-28
-35
GGGCGCC CCACGCC
TATA(AT)AA(GA)
YYAN(TA)YY
(AG)G(AT)(CT)(GAC)
Y C or T (pyrimidine)
The transcription complex either recruits RNA Pol
II or activates a bound RNA Pol II
For review see Smale and Katonga, Ann. Rev.
Biochem. 72 449-479 (2003)
21
Many enhancer elements often lie upstream of
promoters,allowing for many combinations of TF
binding
22
Put a DNA regulatory region upstream of a
reporter gene to analyze its elements
Space for res. enz. to bind
Reportergene
PCR
23
Popular reporters to study promoter/enhancers

• Beta-galactosidase (B-gal) detection by several
  different assays
• Chloramphenicol acetyl transferees (CAT)
  detection, sensitive radioactive assay
• Luciferase (firefly, Renilla jellyfish)
  detection, easy dual, sensitive luminescent
  assay
• Green fluorescent protein (GFP, BFP, YFP))
  cytological, visible in living cells, fusion
  proteins, FACS
• Neomycin phosphotransferase (neo)selectable drug
  resistance (G418R)
• Dihydrofolate reductase (DHFR) selectable in
  dhfr- cells, amplifiable, fusion proteins work

FACS fluorescence-activated cell sorter
24
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
25
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.

26
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.

27

• Got this far

28
Mapping transcriptional elements upstream of a
promoter Mapping with restrictionenzyme
mediated deletions
Conlcusion
29
Footprinting detects sites on DNA to which
protein are bound
DNA DNA-binding protein
Naked DNA
Population of molecules
Population of molecules
missing
30
Note uneven cleavage of naked DNA by DNase
31
Protein-DNA binding EMSA or gel shift
(EMSA electrophoretic mobility shift assay)
1 2 3 4 5
competitor
(supershift)
(shift)
DNA element
(Even though the hexagon looks like a protein
here)
U. Arizona
32
Gel shifts (EMSA
Protein DNA complexes migrate more slowly than
naked DNA
(competed only by specific probe)
(two molecules of protein bound)
(competed by NON-specific probe)
33
SELEX for an RNA binding protein site
A mixture of NA moelcuels of equal size but
randomsequence
Systematic evolution of ligands by exponential
enrichment
T7 RNA polymerase
RT-PCR reverse transcriptase polymerase chain
reaction
34
Synthetic, range usually 6 to 40-mers
SELEX
(T7 RNA Pol from an embedded T7Pol promoter
(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
35
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.
Nucleic acid degenerate base abbreviations
Consensus
Description