Protein DNA Interactions

Jörg BungertDepartment of Biochemistry and

Molecular Biology

Phone: 352-273-8098Email: jbungert@ufl.edu

Objectives• Know the main factors that contribute to the specificity of protein DNA

interactions: Base Readout and Shape Readout.

• Know the major DNA binding motifs in proteins and how they interact with DNA: Helix-Turn-Helix, Zinc Finger, Leucine Zipper.

• Know the difference between DNA bend and DNA kink.

• Understand the consequence of minor groove narrowing.

• Know different methods for analyzing protein DNA interactions: EMSA, ChIP, Selex, Yeast one Hybrid.

Reading: Lodish 7th edition, chapter 7 (pp. 305-315).

Sliding

IntersegmentTransfer

InterdomainAssociation

InterdomainDissociation

Operator

Repressor

How do sequence-specific DNA binding proteins find their target sites in the genome?

Early work by Art Riggs and others have shown that bacterial transcription factors associate

with their respective binding site in solution much faster than predicted according to

macromolecular interaction kinetics. Binding to the specific target involves hydrogen bonding

between the amino acid residues in the active site of the protein and base pairs in the minor

or major grooves of the DNA. Non-specific interactions are electrostatic. Von Hippel and

colleagues proposed that “the DNA cylinder can be viewed as an isopotential surface along

which the protein can diffuse in a one-dimensional random walk”. This sliding mechanism

differs from chemical energy-dependent translocations by e.g. helicases and polymerases.

Spector, Annu. Rev. Biochem., 2003, von Hippel and Berg, JBC, 1989

How do sequence-specific DNA binding proteins find

their target sites in the genome?

Base Readout and Shape Readout

DNA binding proteins combine multiple readout mechanisms to achieve specificity. The topography of the human

genome (assayed by hydroxyl radical cleavage pattern) is evolutionarily constrained and a better predictor of

functional DNA elements than linear DNA. This suggests that DNA topology is an important contributor to the

specificity of protein DNA interactions. Most of the more than 1500 solved protein/DNA structures reveal a form

of bound DNA that deviates from the ideal B-form configuration.

Sequence-Specific DNA interactions

Proteins recognize chemical and conformational signatures of base pairs. Amino acid (AA) residues can interact

with the edges of the base pairs in the major groove. These interactions involve hydrogen bonds, water

mediated hydrogen bonds, or hydrophobic contacts. A:T versus T:A and C:G versus G:C are indistinguishable

in the minor groove. Shown below are rotational views of the dodecamer d(GACT)3 with hydrogen bond donors

and acceptors, thymidine methyl groups, and base carbon hydrogen as indicated. Note that bidentate hydrogen

bonds (two donors and two acceptors) provide more specificity than bifurcate hydrogen bonds (one donor and

two acceptors).

Sequence-Specific Binding Proteins and DNA

1. Each BASE PAIR presents a unique constellation of sites for chemical interaction in the MAJOR GROOVE.

2. A RECOGNITION HELIX on the protein is positioned within themajor groove.

3. The RECOGNITION HELIX participates in hydrogen bonds, Van der Waals interactions, and hydrophobic effect interactions with the base pairs. This is the basis of SEQUENCE RECOGNITION.

4. The most common (>80%) motifs for positioning the recognition helix are the HELIX-TURN-HELIX, the ZINC FINGERand LEUCINE ZIPPER. There are others.

5. Formation of a stable DNA-Protein complex is most often dependentupon additional non-covalent chemical interactions outside of the recognition helix-base pair contacts.

Helix-Turn-Helix

434 RepressorProtein

High resolutionstructure byco-crystallizationwith syntheticoligonucleotides

Homodimer

INDUCED BENDin axis of DNA

Lodish Fig 7-28

Recognition Helix

Protein-Protein

Protein-DNA

The helix turn helix (HTH) motif is the most commonly used secondary structure for specific DNA recognition.

It was first characterized in prokaryotes. A recognition helix is positioned in the major groove and makes base

specific contacts. A second helix stabilizes the recognition helix and is required for the proper positioning.

Eukaryotic homeodomain proteins are characterized by a three helix bundle in which the 2nd and 3rd represent

the HTH. The winged helix-turn-helix (wHTH) motif has an additional b-sheet that interacts with the minor groove

to make additional DNA contacts.

5’: :A-TC-GA-T: :: :: :: :T-AG-CT-A: :

5’

Homodimeric DNABinding Proteins

Homodimeric proteinsspecifically recognize PALINDROMICDNA sequences.Improved specificity

Binding energy additive,so interaction twice asstrong as a monomer.

Some helix-turn-helixproteins are monomericand do not havepalindromic recognitionsequences

Chemical Bonds Between 434R Protein and DNASequence-Specific

Recognition HelixGln28 H-bonds to A (N6 and N7)Gln29 H-bonds to G (O6 and N7)Gln29 and Thr27 van der Waals

contacts with T (CH3)

Sequence-IndependentOutside Recognition Helix

Arg36 in Minor GrooveElectrostatic Interaction

H-bonds peptide backbone and DNA backbone

Branden andTooze Fig 7.17

DNA -- TrpR Repressor ProteinPosition of the Recognition Helix as a mechanism of gene regulation

-- TrpR controls many genes of trp synthesis-- Absence of trp, the TrpR protein is inactivated

-- Trp binds to TrpR altering conformation ofHelix-Turn-Helix motif allowing binding to DNA

Classical Zn-Finger MotifConsensus Sequence:

…C-X5-C-X3-(F/Y)-X5-L-X2-3-H-X3-4-H …

Zinc Finger Proteins may

have more than one Zn

finger per protein.

Zn coordinated by cysteines

and histidines.

Conserved aromatic amino

acid and leucine form a

“strut” positioning the

recognition helix.

Compact 30AA domain, a-helix plus two

antiparallel b-sheets and a Zn2+ ion that is

coordinated by cysteines or histidines.

Recognition Helix

Lodish Fig. 3-9

strut

Zif 268 Recognition HelixInteractions with DNA

Helix PositionZn # -1 2 3 6 DNA

3 Arg Asp Glu Arg GCG

2 Arg Asp His Thr TGG

1 Arg Asp Glu Arg GCG

Zinc Finger Proteins

GL proteinBinds as a Monomer

Five C2H2 Zn fingers

but only four make

sequence-specific

contacts with DNA.

Binding of one helix facilitates the binding

Of the next helix.

Lodish Fig 7-29

Transcription factor TFIIIA and Zn-Finger Encoding Genes

Generation of Zinc Finger Proteins with Altered

Binding Specificity

The way Zn-fingers bind to DNA is well understood. The DNA-sequence

encoding the a-helix reading head can be changed to generate proteins

with altered binding specificity. These altered reading heads can be

assembled into zinc finger proteins to recognize a desired sequence.

An artificial zinc-finger protein with 6 zinc fingers theoretically binds to

a DNA sequence of 18 bp. Thus this protein will bind to one or a few

sequences in the human genome. Artificial Zn-finger DNA binding domains

can be linked to effector domains (transcription activation domain,

transcription repression domain), and expressed in cells to alter expression

of specific genes.

Leucine Zipper Proteins

Homodimeric or Heterodimeric

Recognition Helices:-- extend from zipper helices-- interact with major grooves-- passing on either side of

DNA helical axis

Leucine Zipper Proteins

Homo- or Heterodimeric DNA Binding Proteins

“Leucine Zipper”-- series of about 7 Leu -- along helical faces

of each protein-- hydrophobic (van der

(Waals) interactionsresult in dimerization

RECOGNITION HELIX

Coiled-Coil

Lodish Fig 7-29

Lodish Fig. 4-5

Most DNA binding proteins use an a-helix that

Recognizes bps in the major groove. Some proteins,

like TBP use b-sheets that face into the minor groove.

This mode of interaction often deforms the DNA

leading to sharp bends.

Global Shape Readout: A-B-Z-DNA

Local Shape Readout: Bend, Kink, Narrow Minor Groove

DNA kinks are characterized by a

complete or partial loss of stacking of

a single base pair step. For example

TpA has the weakest stacking

interaction and is the most flexible of

the 10 unique dinucleotides.

(“hinge”). Proteins can stabilize the

kink by intercalation of hydrophobic

side chains. The width of the minor

groove depends on the roll (relative

rotation between adjacent bases with

respect to base pairing axis), helix

twist (relative rotation between

adjacent bases with respect to helix

axis) or propeller twist. ApT base pair

steps have negative roll angels and

compress the minor groove.

Compression of the minor groove

increases the negative electrostatic

potential, which often recruits

arginine residues.

Hox (homeodomain) binding specificity mediated by local shape recognition

Hox DNA-binding specificity mediated by local shape recognition. All panels show either the fkh250-binding site or the fkh250conbinding site. fkh250, but not fkh250con, has two minor groove minima, which creates a more negative electrostatic potential (minus signs). The capital letterW refers to the Hox YPWM motif, which makes a direct contact with the cofactor Exd. (a) In the absence of Exd, Scr does not bind with high affinity to fkh250 because basic side chains (small bars), in particular, arginines, on the N-terminal arm and linker of Scr are not positioned correctly. (b) Other Hox proteins do not bind well to fkh250 even in the presence of Exd because their N-terminal arms and linker regions have different sequences. (c) The Scr-Exd heterodimer binds well to fkh250 because the Scr N-terminal arm and linker region have the correct residues, and Exd positions them correctly by binding the YPWM motif (W). (d ) Other Hox-Exd heterodimers bind well to fkh250con. This binding site is not as selective because it has a less negative electrostatic potential. Thus, the sequences of the Hox N-terminal arms and linker regions are not as important for binding.

End-labeledoligonucleotide

probe

Probe + Nuclear Extract

Probe + Nuclear Extract

+ Antibody1 2 3

Autoradiogram of EMSA Acrylamide

Gel

Electrophoretic Mobility Shift Assay (EMSA)

“Band Shift”

“Supershift”

HIGHEST MOBILITY

LEAST MOBILITY

Is a protein present that binds a DNA sequence?Does a specific known protein binding a sequence?An in vitro method.

EMSA Gel Autoradiogram

Purified DNA Binding Domain of the Glucocorticoid Receptor (GR)Probe oligonucleotide contains a GR binding site from the Pal gene

Band shift

Probe

Meijsing et al (2009) Science 324, 407 Suppl.

Chromatin Immunoprecipitation Assay (ChIP)

(Protein A sepharose)

Formaldehydetreated cells

(Sonifier)

PCR using gene-specific oligonucleotide primers

PCR products detected byagarose gel electrophoresis Adapted from Lodish Fig 7-37

Does a known protein bindto a specific DNAelement in the cell?

An in vivo approach.

Mechanically shear DNAAdd antibody specific for a DNA Binding Protein

Antibody

Methods for studying DNA-protein interactions

B. Protein-binding microarray

(PBM). All possible 10-base-long

sequences are included on the

array. Primer directed DNA

synthesis creates dsDNA. After

binding of the protein, fluorescently

labeled anitbodies are used for

detection.

C. High-Throughput (HT)-SELEX.

DNA molecules from a random

library are exposed immobilized

transcription factor (TF), unbound

DNA is washed off, and bound DNA

is subjected to sequencing.

D. Bacterial one-hybrid (B1H)

system. The TF is fused to a subunit

RNA polymerase. A sequence from

a randomized library is inserted

upstream of the promoter of the

HIS3 gene. When the TF binds to

the randomized sequence, the HIS3

promoter becomes more active,

which increases the growth rate.

Stormo and Zhao, Nat. Rev. Genetics, 2010,

Stormo and Zhao, Nat. Rev. Genetics, 2010,

Affinity: Binding of protein

to DNA is a bimolecular

process governed by two

rates: on-rate (formation of

complex), and off-rate

(dissociation).

TF + S TF.S

The Kd can be calculated

from the concentration of

the product (TF.S) divided

by the concentration of

protein (TF) and sequence

(S). The sequence binding

probability (PS bound) is a

reflection of Kd and TF

concentration

(concentration of TF

divided by concentration

of TF plus Kd.).

The graph on the bottom

depicts the binding

propabilties of many

different sequences

dependent on TF

concentration.

Affinity and Consensus Sequence

Position Weight Matrix (PWM): a score is assigned

to each possible base at each position. The sum

of the elements that correspond to a specific

sequence gives a total score for this sequence. The

„logo‟ provides a convenient graphical representation

of the PWM.

Distinct dimensions of conservation to consider. There are many ways to assess the conservation of an

observed binding event. In this cartoon example, we consider a binding event in humans (shown in green) to

the orthologous region in mice. (a) The sequence under the binding event can be conserved to mice. (b) If in

vivo binding data are available in mice, we can ascertain (i) when a binding event is species-specific or (ii)

when it is observed in both species. (c) By assigning binding events to nearby genes, we also observe cases of

turnover, where the target of a binding event is maintained. In this case, the loss of one binding event is

compensated by a nearby gain, conserving the gene as a target of the transcription factor.

Transcription factor–DNA interactions.

(a) A transcription factor recognizes a

DNA sequence motif, shown here as a

logo representation of a PSSM where the

height of each letter is proportional to the

score of the nucleotide at that position of

the motif. (b) The location of protein–DNA

interactions can be assayed by ChIP. The

magnitude of the ChIP enrichment signal

(shown in green) correlates broadly with

different levels of transcription factor

occupancy [30] , [31] , [57] and [58] .

Given the ChIP data, one can assess the

relationship between occupancy and

sequence: (i) not all high-affinity

recognition motifs (shown as purple

boxes) are bound, (ii) some binding

events do not have a high-affinity motif

associated (but nearly all sequences

contain low-affinity sites) and (iii) some

binding events are at high-affinity motifs

Transcription factor binding variation in the evolution of gene regulation

Dowell, Trends in Genetics, 2010