How DNA Binding Proteins Find Their DNA Target Sites

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How DNA Binding Proteins Find Their DNA Target
Sites
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• 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)

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Rpol has general/weak affinity for normal B-form
DNA

• For Rpol to find promoter it must
• Dissociate from site 1Find 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

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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
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Rpol searches are NOT diffusion limited
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• 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.

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• Searching much faster
• WHY?
• - Association/dissociation virtually
  simultaneous
• - NO time wasted commuting between sites

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Rpol binds VERY rapidly to random DNA sites
Could find promoter by direct displacement of
bound sequence
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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

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What Drives intersegment transfer of DBP in the
search mode?
ENTROPY
HOW?
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Search is entropically driven

• FIRST DNA has an ion atmosphere rich in
  counterions depleted in co-ions

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Ligand binds DNA
DPBx



Release of Z counterions upon binding creates
disorder entropy This is a favorable reaction
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Ligand binds DNA
Moves
Rebinds
Ptn exchanges to new site Counterions rearrange
back to ion cloud Upon binding to new contact
site, counterions in cloud get redistributed
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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.

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Reaction
Add Counterions
Dissociate
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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)

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EMSA

• The EMSA technique proteinDNA 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 proteinDNA 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

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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
• 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.

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Critical EMSA Reaction Parameters
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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).

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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 RNAprotein
  interactions and peptideprotein interactions.

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Non-Specific Competitor DNA


Limiting
Excess
Nonspecific competitor DNA poly(dIdC) or
poly(dAdT) 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
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Real Data

• 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

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Competition Expt
Heterologous cold DNA
Complex amount
Homologous probe cold probe
DNA Concentration
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Other EMSA Applications

• Supershift Reactions To identify ligand and DNA
• Antibody Binds ligand in complex and
  supershifts
• Antibody may disrupt the proteinDNA 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/

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AB
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Binding Reaction Components

• Factors that affect the strength and specificity
  of the proteinDNA 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.

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Ionic strength
Add Counterions
Dissociate
Usually Keep ionic strength (total z)
LOW. Note Preparing a crude extract from
nuclei, requires HIGH SALT EXTRACTS WHY?
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Recap Effects of Ionic Strength on DNA-protein
interactions

• DNA L DNA-L Z

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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
Nucleus
NaCl (0.5 M)
Free Proteins
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• 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

Specific Site
Released Z
Sliding over non specific DNA sites leads to
specific site with long half life
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• Important take home messages on DNA binding
  proteins

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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!

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Heterologous DNA (E. coli or salmon sperm or poly
dIdC competitor)
DNA complex amount
Same DNA as probe (set at 2 ng/assay) self
competition
DNA-Ptn complex
Free probe
DNA Concentration
5000 ng or 5 ug
5 ng
Assay conditions 32P DNA Fragment (200 bp) _at_
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)
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Other ways to examine DNA binding proteins at
cognate sites
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DNase Footprints
Must also include non-specific binding DNA along
with target
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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)

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Early Evidence of DNA bending which enhances ptn
access
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SPLICING
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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
  lt10)

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RNA Splicing

• Primary transcript pre-mRNA
• Must be processed
• Splicing converts pre-mRNA to mRNA
• Alternative splicing can increase gene diversity
• Estimated 60 of genes are alt. spliced!
• One gene could encode 1000s of splice variants!
• Accuracy is CRITICAL, mistakes not tolerated

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Mechanisms

• Consensus sequences in the transcript are key to
  precise splicing outcomes

Consensus site _at_ splice junctions HIGHLY
conserved especially GU and AG
Branch point mid intronnear poly Pyr tract
Donor site
Acceptor Site
NOTE THAT THE CONSENSUS ELEMENTS ARE IN INTRONS
AND NOT EXONS (CONSTRAINED BY CODING SEQUENCE)
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Intron excision involves formation of a lariat
structure

• Splicing is a continuum
• 2 successive transesterifications
• Phosphodiester linkages break/reseal in a coupled
  reaction
• Rxn can be visualized as a 2-step process
• 1st is 2OH at conserved A residue
• 2nd is formation of lariat and splice product

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Nucleophilic attack _at_ P
Result Freed 5 end of intron joins A to make
the branch site in lariat
1st rxn
3 way junction2 OH Link at A
Nucleophilic attack _at_ P in splice site junction
2nd rxn
2 Products are made as a result
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Key points

• No net increase in phosphodiester bonds
• 2 bonds are broke and 2 are made
• No energy input required in transesterification
  reactions
• However, ATP is consumed
• Required for maintenance/assembly of splicing
  machinery in vivo

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If no net energy input, what makes splicing
reaction irreversible?

• Entropically driven by
• Breaking a single RNA transcript in two creates
  disorder (favorable)
• Rearrangement of ion clouds in process
• Exicised intron rapidly degraded
• Thus, cannot go back or reverse the splicing
  reaction

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Trans-splicing

• Exons from different transcripts are fused
• Rare in animals but does occur
• More common in C. elegans, trypanosomes

No lariat a Y structure is formed instead
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Splicesomes

• Large complexes or molecular machines carry out
  splicing in vivo

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Splicing machines RNPs

• gt150 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

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snRNP Roles

• Recognize 5 splice site and branch site
• Bring these sites into proximity
• Catalyze the splicing reaction

Discuss in detail
RNA-RNA RNA-protein Protein-Protein
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• Different snRNPs recognize same (or overlapping)
  sites in transcript
• Here U1 and U6 shown to bind to splice site
  (donor)

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• snRNP U2 binds branch site

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• RNA pairing between snRNP U2 amd U6 is shown
• Brings 5 splice site and branch site into
  proximity

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Branch point binding protein

• Here BBP (not part of splicesome) recognizes A
  region and is displaced by U2 during the reaction
  sequence

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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

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• Mechanistic overview
• U1 snRNP binds 5 splice site
• U2AF binds Pyr tract and 3 splice site (U2AF has
  2 subunits)
• U2AF interacts with BBP to help stabilize this
  interaction
• U2 snRNA binds A branch site and displaces BBP
  A complex
• A residue extrudes and made available to bond w.
  5 splice site
• A complex reorganized to bring together all 3
  splice sites
• U4 and U6 snRNAs along with U5 join to form the
  tri-snRNP complex
• Entry of tri-snurp complex defines formation of
  B complex
• 7. U1 exits and is replaced by U6 ( C complex)
  or active site.

A complex
B complex
U4 exits and U2 takes over to complete
order not well known
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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

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Nuclear pre-mRNA

• Abundance
• Very common used in most eukarya
• Mechanism
• Transesterifications branch A site
• Catalytic mechanism
• Major spliceosome

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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)

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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

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Group I Introns Release Linears

• Different pathway to splicing
• Uses free G (not branch _at_ A)
• G residue bound to RNA and its 3OH presented to
  splice site.
• Gp I introns have an internal guide sequence that
  pairs with 5 splice site
• Directs nucleophilic site of G attack

G binding pocket forms on RNA
free 3 end of exon attacks 3 splice site
RIBOZYMES
linear byproduct
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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.

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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)