How DNA Binding Proteins
Find Their DNA Target Sites
•Rpol distribution in
cell (in vivo)
•Core and
holoenzyme are all
thought to be DNA
bound
•VERY little is free
•Excess core is in
loose complexes
(scanning)
Rpol has general/weak affinity for normal B-form DNA
• For Rpol to find promoter it must:
– Dissociate from site 1;Find site 2
– Bind site 2
• Movement of Rpol is DIFFUSION LIMITED (for a 60 bp
site rate constant MUST be less than
10-8M-1sec-1 (max diffusion rate for a molecule to move
through medium is less than 10-8M-1sec-1)
• Actual rate in vitro is greater than this (or equal to this
value).
• If this applies in vivo: time required for successive cycles
of dissoc/assoc. is too great to account for txn responses
Conceptually: Holoenzyme must release and rebind to
find promoter.
The rate is limited by diffusion; ie, how fast a
macromolecule can migrate at random through a
physiological solution at 37oC.
BUT…. This process is MUCH MUCH faster!
Thus: Diffusion cannot explain how Rpol finds a target
promoter inside the cell
• Rpol locating binding sites.
– Significantly speeded up: if the initial target for RNA
polymerase is the whole genome,
– Not just a specific promoter sequence.
– By increasing the target size (genome) rate constant
for diffusion to DNA increases
– No longer limiting.
• MODEL: one bound sequence directly displaced
by another sequence.
– Thus, enzyme exchanges one sequence with another
sequence very rapidly
– Continues to exchange sequences until a promoter is
found.
• Searching much faster
WHY?
- Association/dissociation
virtually simultaneous
- NO time wasted „commuting‟
between sites
Rpol binds VERY
rapidly to random
DNA sites
Could find promoter
by direct
displacement of
bound sequence
Protein exchange of DBP
(DNA binding proteins)
• Could be linear diffusion
• Could be 3-D intersegment transfer
• Most probably 3-D transfer
• Important point:
All sequence specific DNA binding
proteins bind DNA in a non-specific
(non-seq) dependent mode first.
• This initiates the search for specific site
What Drives intersegment transfer
of DBP in the search mode?
Search is entropically driven
• FIRST: DNA has an ion atmosphere rich
in counterions; depleted in co-ions
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
Ca
3-4
[Counter ion]
Molar
Distance from helix
Ligand binds DNA
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
DPBx
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++
+++++++
+++++++
Release of Z+ counterions upon
binding creates disorder = entropy
This is a favorable reaction
Ligand binds DNA
Rebinds
„Moves‟
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++
+++++++
+++++++
+++++++
+++++++
Ptn exchanges to new site: Counterions rearrange
back to ion cloud
Upon binding to new contact site, counterions in
cloud get redistributed
Ligand finds DNA specific
sequence
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
DPBx
+++++++++++++++++++++++++++++++++++++++++++++
+++++++++++++++++++++++++++++++++++++++++++++
+++++++
+++++++
+++++++
• Rapid exchange between sites stops when DPBx finds a
high affinity, sequence specific site it „likes‟
• Usually involves base specific contacts that either alter
structure of protein or (more likely) bring specific domains
of ligand into play at DNA target sequence.
Reaction
DNA + Ligand DNA-Ligand + Z+
METHOD: How one finds DBPs?
• Goal: Find whether a protein binds a
specific sequence you believe is
regulatory site
• You have a 10 bp sequence (in a 100 bp
fragment
• Carry out Electrophoretic Mobility Shift
Assay (EMSA)
• The EMSA technique: protein:DNA complexes migrate
more slowly than free DNA in non-denaturing gel
electrophoresis (= low ionic strength gels)
• Complexes Shift (retarded) upon protein binding:
assay also referred to as a gel shift or gel retardation
assay.
• Early expts on protein:DNA interactions: primarily
used nitrocellulose filter-binding assays
• Advantages of EMSA
– resolves complexes of different stoichiometry (and
conformation).
– Works with crude extracts & purified preparations
– Can be used in conjunction with mutagenesis: identify key
binding sequence in any regulatory region.
– EMSAs can also be utilized quantitatively to measure
thermodynamic and kinetic parameters.
– Combined with antibodies to characterize specificity
EMSA
• Ability to resolve complexes depends on stability of
the complex during the brief time (approximately 30
minutes) it is migrating into the gel.
• Sequence-specific interactions are stabilized by low
ionic strength
DNA + Ligand DNA-Ligand + Z+
• Upon entry into the gel, proteins quickly resolved
from free DNA
– “Freezing” the equilibrium between bound and free DNA.
– In the gel, the complex may be stabilized by “caging” effects
of the gel matrix, meaning that if the complex dissociates,
its localized concentration remains high, promoting prompt
reassociation.
– Even labile complexes can often be resolved by this
method.
Critical EMSA Reaction Parameters
Target DNA (=probe)
• Linear DNA fragments containing binding
sequence(s) used in EMSAs.
• Labeling Probe:
– 5‟ end label with g-[32P]-ATP and polynucleotide
kinase
– 3‟ end label with Fill-in reactions a-[32P]- dXTP.
– Need to have high specific activity probe (at least 1 x
106 cpm/ug)
– EMSA binding expts use about 5 -10 ng of DNA
probe (ca. 10,000 cpm)
– Non-Radioactive detection: DNA biotinylated then
probe with chemiluminescent substrate.
• If the target DNA is short (20-50 bp) oligo
bearing the specific sequence work well
(annealed to form a duplex).
Target DNA (=probe)
• Some DNA/ptn complexes involve multiprotein
complexes
– Requires multiple proteins and often longer DNA fragments to
accommodate multiprotein complexes
– Larger DNA probes (100-500 bp): a restriction fragment or PCR
product is used to prepare probe
• DNA/Ptn complexes result in retarded mobility in the gel.
– Circular DNA probes (e.g., minicircles of 200-400 bp):
complexes may migrate faster than the free DNA.
• Gel shift assays are also good for resolving altered or
bent DNA conformations that result from the binding of
certain protein factors.
• Gel shift assays work with RNA:protein interactions and
peptide:protein interactions.
Non-Specific Competitor DNA
DNAns + L DNAns-L + DNAs DNAs-L + DNAns
* *
Excess Limiting
Nonspecific competitor DNA: poly(dI•dC) or poly(dA•dT) minimizes binding
of nonspecific proteins to the labeled target DNA.
These repetitive polymers do the following:
-provide an excess of nonspecific sites to adsorb proteins in crude lysates
that will bind to any general DNA sequence.
-provide a 3-D intersegment transfer structure for the specific DBP to act
Non-competitor is usually present in 100-1000 fold excess:
Example: 10 ng of labeled probe + 1000-5000ng ng of cold competitor
Real Data
Lane 1 2 3 4
EBNA Extract - + + +
Unlabeled EBNA
- - + -
DNA
Unlabeled Oct-1
- - - +
DNA
Shows self competition:
• Rxn contains 1 -2 ng of EBNA DNA probe (32P
Label) and 1 ug polydI-dC cold competitor.
• Self competition in lane 3: added 2 ng of cold
EBNA DNA (loss of complex)
• Adding 2 ng of heterologous DNA (Oct-1): no
dissociation
Competition Expt
Heterologous cold DNA
Complex [amount]
Homologous
probe cold
probe
DNA Concentration
Other EMSA Applications
• Supershift Reactions: To identify ligand and DNA
• Antibody: Binds ligand in complex and supershifts
• Antibody may disrupt the protein:DNA interaction
– Proper controls will reveal such “negative” results.
• Supershifts could include other secondary or indirectly
bound proteins as well.
– An alternative identification process would be to perform a
combination “Shift-Western blot.”
– Transfer complexes to stacked nitrocellulose and anion
exchange membranes as blots.
– Blot probed with a specific antibody (Westerm) while
autoradiography or chemiluminescent techniques can detect the
DNA captured on the anion-exchange membrane/
Extract - + + +
Antibody - - + -
Competitor - - - +
AB
Binding Reaction Components
• Factors that affect the strength and specificity of the protein:DNA
interactions
• Ionic strength
• pH
• Nonionic detergents, glycerol or carrier proteins (e.g., BSA),
• Divalent cations (e.g., Mg2+ or Zn2+)
• Concentration and type of competitor DNA present,
• Temperature and time of the binding reaction.
• If a particular ion, pH or other molecule is critical to complex
formation in the binding reaction, it is often included in the
electrophoresis buffer to stabilize the interaction prior to its entrance
into the gel matrix.
Ionic strength
DNA + Ligand DNA-Ligand + Z+
Usually: Keep ionic strength (total [z+]) LOW.
Note: Preparing a crude extract from nuclei, requires HIGH SALT EXTRACTS
WHY?
Recap:
Effects of Ionic Strength on DNA-
protein interactions
DNA + L DNA-L + Z+
+ +
+
+ +
+ +
++++++++++++++++++++ ++++++++++++++++++++
++++++++++++++++++++ ++++++++++++++++++++
Role of Z+ ions, DNA Ion atmosphere, and non-
specific DNA complexes vs. specific DNA
complexes explains how DBPs can be extracted
and assayed by gel shifts and DNase I foot printing
• Effect of ionic strength: high salt is important
to extract DBPs from nucleus for biochemical
analyses
Free DNA
+NaCl (0.5 M)
Nucleus
Free Proteins
• Importance of non-specific binding: to reduce
dimensionality of search; defines why non-
specific competitor DNA must be used in gel
shift assays.
– Half life of non-specific complexes very short while
specific complexes have much longer half lives
Released Z+ Specific Site
Sliding over non specific
DNA sites leads to specific
site with long half life
Important take home messages
on DNA binding proteins
Assays for DBP must take non-specific
binding parameters into account
• Gel shift assays: in theory very simple: low ionic strength (10
mM) PAGE. Encourages DNA binding complexes.
• Must always include a probe (32P label) present in small
amounts (10 ng) PLUS excess non-specific DNA (1-10 ug).
Why? The T1/2 of the specific complex is MUCH longer than
the non-specific one. This is critical to include to document
specificity!
Heterologous DNA (E. coli or salmon sperm or poly dIdC competitor)
DNA Competitor
DNA Competitor
DNA Compete
+DBP-X +5 ug
+DBP-X +5 ng
+DBP-X no
Ref
DNA complex amount
Origin
DNA-Ptn complex
Free probe
DNA Concentration
5 ng 5000 ng
or 5 ug
Assay conditions:
32P DNA Fragment (200 bp) @ 106 cpm/ug (2000 cpm or 2ng)
Shows that self competition of a DNA protein complex is SPECIFC and that you are
detecting a sequence specific DNA binding event (with a 32P probe)
Other ways to examine DNA
binding proteins at cognate sites
DNase Footprints
Must also include non-specific
binding DNA along with target
Key points
• All DNA site specific binding proteins have a
general affinity for DNA that is weak and a
necessary precursor to specific site binding.
• There is a strong ionic strength dependence of
DNA binding (for both modes)
• Gel shifts and footprinting expts. With DBPs
requires judicious knowledge of ionic strength
(usually low) and of appropriate amount of
competitor DNA (usually in huge excess over
target probe).
• DNA binding can be enhanced by alterations in
DNA structure (like DNA bending)
Early Evidence of DNA bending which enhances ptn access
Rate: 40nt/sec
Poly A, 5’ cap
• Eukaryotic genes are mosaics of Int (non coding) and
Exons (coding)
• Exons typically small (150 bp average)
• Introns: can be small or huge and MANY
– DHFR Gene: 31 kb, 6 exons, 2 kb mRNA (coding DNA 150 proteins
• 5 RNAs
– Small nuclear RNAs (snRNAs): U1,2,4,5,6
– Ca. 100 and 300 nt long complexed with
protein (snRNP or snurps)
– RNPs and misc. ptns come and go in process
• Process mediated primarily by RNA
catalysis with protein support
• Akin to a ribosome
snRNP Roles
1. Recognize 5‟ splice site and branch site
2. Bring these sites into proximity
3. Catalyze the splicing reaction
Discuss
in detail
• Different snRNPs recognize same (or
overlapping) sites in transcript
• Here: U1 and U6 shown to bind to splice
site (donor)
• snRNP U2 binds branch site
• RNA pairing between snRNP U2 amd U6
is shown
•Brings 5‟ splice site and branch site into
proximity
Branch point binding protein
• Here: BBP (not part of splicesome)
recognizes A region and is displaced by
U2 during the reaction sequence
Other protein roles
• U2AF: binds poly-pyr tract; helps BBP
bind to branch
• RNA-annealing factors
– Help load snRNPs onto transcript
• DEAD Box helicases
– Use ATPase to dissociate RNA duplexes
– Facilitate alternative RNA-RNA interactions
Mechanistic overview
1. U1 snRNP binds 5‟ splice site
2. U2AF binds Pyr tract and 3‟ splice
A complex site (U2AF has 2 subunits)
3. U2AF interacts with BBP to help
stabilize this interaction
4. U2 snRNA binds A branch site and
B complex
displaces BBP = “A complex”
5. A residue extrudes and made
available to bond w. 5‟ splice site
6. A complex reorganized to bring
together all 3 splice sites:
U4 exits and
1. U4 and U6 snRNAs along with U5 join
U2 takes
over to to form the „tri-snRNP complex‟
complete 2. Entry of tri-snurp complex defines
formation of “B complex”
7. U1 exits and is replaced by U6 (= C
complex) or active site.
How did splicing evolve?
• Its complicated… lots of players
• Probably evolved from self splicing
mechanisms with catalytic RNA
• Summary of 3 classes of RNA Splicing
Nuclear pre-mRNA
• Abundance:
– Very common; used in most eukarya
• Mechanism:
– Transesterifications; branch A site
• Catalytic mechanism:
– Major spliceosome
Group II Introns
• Abundance:
– Rare; some eukaryotic genes from organelles
– Prokaryotic mechanism
• Mechanism:
– Transesterifications; branch A site
• Catalytic mechanism:
– RNA encoded by intron (= Ribozyme
mediated)
Group I Introns
• Abundance:
– Rare; nuclear rRNA in some eukaryotes
– Organelles genes
– A few prokaryotic genes
• Mechanism:
– Transesterifications; branch G site
• Catalytic mechanism:
– RNA encoded by intron (= Ribozyme mediated)
– NOTE: Not a true enzyme catalytic event! [mediate
only one round of events]
Group I Introns Release Linears
• Different pathway to splicing G binding pocket
forms on RNA
• Uses free G (not branch @ A)
– G residue bound to RNA and its „free‟ 3‟ end of
exon attacks 3‟
3‟OH presented to splice site. splice site
• Gp I introns have an internal
guide sequence that pairs
with 5‟ splice site
– Directs nucleophilic site of G
attack
= linear byproduct
Gp I Introns can act as ribozymes
• Provide free G in excess
(there is a terminal G at 3‟ end
of intron)
• Any RNA with homology to
Internal guide seq. (IGS) will
be degraded
• By modifying IGS, we can
target specific mRNAs for
degradation
• Thereby modulate gene
expression in cells.
Gp I introns: Most of the RNA
essential for self-splicing reactions
• Usually 400-1000 nt long
• Most or all essential
– Because folding of RNA is especially critical
• In vivo: ptn factors important in stabilizing
proper configuration of RNA backbone
• In vitro: VERY high salt concentrations
can compensate (self-splicing rxns can
occur in vitro)