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How DNA Binding Proteins Find Their DNA Target Sites

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Core and holoenzyme are all thought to be DNA bound. VERY little is free ... an excess of nonspecific sites to adsorb proteins in crude lysates that will ... – PowerPoint PPT presentation

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Title: How DNA Binding Proteins Find Their DNA Target Sites


1
How DNA Binding Proteins Find Their DNA Target
Sites
2
  • 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)

3
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

4
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
5
Rpol searches are NOT diffusion limited
6
  • 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.

7
  • Searching much faster
  • WHY?
  • - Association/dissociation virtually
    simultaneous
  • - NO time wasted commuting between sites

8
Rpol binds VERY rapidly to random DNA sites
Could find promoter by direct displacement of
bound sequence
9
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

10
What Drives intersegment transfer of DBP in the
search mode?
ENTROPY
HOW?
11
Search is entropically driven
  • FIRST DNA has an ion atmosphere rich in
    counterions depleted in co-ions

12
Ligand binds DNA
DPBx



Release of Z counterions upon binding creates
disorder entropy This is a favorable reaction
13
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
14
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.

15
Reaction
Add Counterions
Dissociate
16
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)

17
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

18
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.

19
Critical EMSA Reaction Parameters
20
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).

21
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.

22
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
23
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

24
Competition Expt
Heterologous cold DNA
Complex amount
Homologous probe cold probe
DNA Concentration
25
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/

26
AB
27
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.

28
Ionic strength
Add Counterions
Dissociate
Usually Keep ionic strength (total z)
LOW. Note Preparing a crude extract from
nuclei, requires HIGH SALT EXTRACTS WHY?
29
Recap Effects of Ionic Strength on DNA-protein
interactions
  • DNA L DNA-L Z

30
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
31
  • 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
32
  • Important take home messages on DNA binding
    proteins

33
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!

34
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)
35
Other ways to examine DNA binding proteins at
cognate sites
36
DNase Footprints
Must also include non-specific binding DNA along
with target
37
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)

38
Early Evidence of DNA bending which enhances ptn
access
39
SPLICING
40
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)

41
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

42
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)
43
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

44
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
45
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

46
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

47
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
48
Splicesomes
  • Large complexes or molecular machines carry out
    splicing in vivo

49
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

50
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
51
  • Different snRNPs recognize same (or overlapping)
    sites in transcript
  • Here U1 and U6 shown to bind to splice site
    (donor)

52
  • snRNP U2 binds branch site

53
  • RNA pairing between snRNP U2 amd U6 is shown
  • Brings 5 splice site and branch site into
    proximity

54
Branch point binding protein
  • Here BBP (not part of splicesome) recognizes A
    region and is displaced by U2 during the reaction
    sequence

55
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

56
  • 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
57
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

58
Nuclear pre-mRNA
  • Abundance
  • Very common used in most eukarya
  • Mechanism
  • Transesterifications branch A site
  • Catalytic mechanism
  • Major spliceosome

59
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)

60
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

61
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
62
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.

63
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)
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