Nucleic Acid Interaction

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Nucleic Acid Interaction

• Noncovalent interaction with
• Large molecules (Proteins)

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Nucleic Acids interact reversibly with
Major Groove
Water Metal ions Small organic
molecules Drugs Carcinogens Antibiotics Protein
s
Minor Groove
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Forces between proteins and DNA
Electrostatic Salt bridges Dipolar Hydrogen
bonds Entropic The hydrophobic
effect Dispersion base stacking
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Forces between proteins and DNA
Electrostatic Salt bridges Interaction between
groups of opposite chargeOccur between the
ionized phosphates of the nucleic acid and either
thee-amino group of lysine, the guanidinium
group of arginine, or the protonated imidazole
of histidine.
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Forces between proteins and DNA
Dipolar Hydrogen bonds d- d d-
dX H ----- Y R X and Y are nitrogen and
oxygen in biological systems Positioning of
hydrogen bond donors (X) and acceptors (Y) is
optimized between protein and DNA.
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Forces between proteins and DNA
Entropic The hydrophobic effect A complementary
surface formed between a protein and anucleic
acid will release ordered water molecules at the
surface ofthe protein or nucleic acid. The
formerly ordered water molecules become part of
the disordered bulk water, thus stabilizing the
interaction through an increase in the entropy
of the system. Consequently, the surfaces of the
protein and nucleic acid tend tobe exactly
complementary, increasing the specificity of the
interaction.
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Forces between proteins and DNA
Dispersion base stacking Base stacking is
dependent on the hydrophobic effect aswell as
dispersion (London) forces. Molecules with no net
dipole can attract each other by a
transientdipole-induced dipole effect. These
forces are weak but do play a role in protein
nucleic acid interaction,specifically in base
stacking.
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Protein Review
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Amino Acids
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Amino acids with aliphatic side chains
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Amino acids with aromatic side chains
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Amino acids with polar, uncharged side chains
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Amino acids with positively charged side chanis
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Amino acids with negatively charged side chains
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Alpha helix
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Alpha helix
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Alpha helix
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Beta sheet
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Beta sheet
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Interaction between proteins and double-stranded
B-DNA
Since B-DNA has a net negative charge, protein
domains that interact with B-DNA often have a net
positive charge. The proteins may have polar or
charged side chains that interact with the
phosphate oxygens of the B-DNA backbone.
Approximately half of the protein-DNA
interactions are with the DNA backbone.
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E. coli DNA polymerase III has a doughnut-shaped
hole linedwith positively-charged amino acid
side chains thatinteract with the
negatively-charged DNA strand
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Interaction between proteins and double-stranded
B-DNA
Since B-DNA has a net negative charge, protein
domains that interact with B-DNA have a net
positive charge. The proteins have polar or
charged sidechains that interact with the
phosphate oxygens of the B-DNA backbone.
Approximately half of the protein-DNA
interactions are with the DNA backbone. Many
DNA-binding proteins have an alpha helix that
interacts with nucleotide bases in the major
groove. An amine-containing amino acid side
chain often forms hydrogen bonds with major
groove bases.A single amino acid may form
hydrogen bonds with multiple, adjacent nucleotide
bases, increasing sequence-specific
interaction.Common amino acid arginine or
glutamine
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The catabolite activator protein(CAP) from E.
coli uses alphahelices to interact with
nucleotide bases in the majorgroove of the DNA
helix.
a-helix
a-helix
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Interaction between proteins and single-stranded
DNA
During certain cellular processes, DNA will
become single-stranded.Example Replication of
DNA Proteins are important for replication and
must recognize and bind to single-stranded DNA.
The single-stranded DNA often have the
nucleotide bases exposed, which interact with
hydrophobic or aromatic amino acid side chains of
proteins. The proteins will also have a region
of net positive charge for interaction with the
phosphate backbone of the DNA.
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The kinetics of forming protein DNA complexes
Proteins often bind to specific sequences of
DNA.Example Restriction enzyme EcoRI binds to
the DNA sequence5-GAATTC-33-CTTAAG-5
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EcoRI bound to DNA
C
T
T
A
A
G
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The kinetics of forming protein DNA complexes
Proteins often bind to specific sequences of
DNA.Example Restriction enzyme EcoRI binds to
the DNA sequence5-GAATTC-33-CTTAAG-5 How
do proteins find their target DNA sequence? 1.
Randomly bind, dissociate, re-bind until they
find their sequence?(Three-dimensional random
walk) 2. Bind non-specifically and then slide
along DNA until they find it?(One-dimensional
walk)
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Non-sequence specific protein DNA interaction
From the moment a new strand of DNA is
synthesized to the moment it is degraded in a
cell, there are proteins associated with it. Many
of these proteins interact in a non-sequence
specific manner.Many of the proteins are
involved in packaging the DNA. Example histone
proteins that form the nucleosome Proteins that
interact non-specifically with DNA interact with
the negatively-charged ribose-phosphate backbone.
Therefore, they have a high percentage of basic
amino acid side chains such as lysine and
arginine.
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Nucleosomes
DNA in eukaryotic cells is packaged into
nucleosomes,which contain proteins called
histones.
DNA wrapped around a histone core (side view)
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DNA polymerase III
Phosphodiester bonds are formed by a nucleophilic
attack of a free 3-OH on the 5-a-PO4 of the
incoming dNTP. This reaction is catalyzed in E.
coli by DNA polymerase III
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Requirements for DNA replication
The enzyme DNA polymerase. A DNA template to
guide synthesis. A primer, a short nucleotide
segment complementary to the template that can
provide a free 3-OH for synthesis. Primers are
often short RNA oligonucleotides
synthesized when and where needed by specialized
enzymes.
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E. coli DNA polymerase III has a doughnut-shaped
hole linedwith positively-charged amino acid
side chains thatinteract with the
negatively-charged DNA strand
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E. coli DNA polymerase III makes no
sequence-specificcontacts instead it makes
hydrogen bonds to the sugar-phosphate backbone,
and hydrophobic and base-stacking contacts to the
nucleotide bases
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DNA polymerase III
Annual Review of Biochemistry (1995) 64, 176-200
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Specific protein DNA interactions
For a cell to function, proteins must distinguish
one nucleic acid sequence from another very
accurately. Activators and repressors of
transcriptionturn specific genes on and
off. Common themes of protein - DNA
interaction 1. Helix-turn-helix 2. Zinc
finger 3. Leucine zipper
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Helix-turn-helix motif
Often found in proteins that regulate gene
transcription. The pair of a-helices stack to
form a V shape with an angle of about 60º The
first helix positions the second helix. The
second helix binds to the DNA, projecting into
the major groove and recognizing specific
sequences.
Shown is ahelix-turn-helix motiffrom a
homeodomain protein, A family of proteins
thatbinds to eukaryotic DNAand regulate
transcriptionof specific genes.
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Zinc finger motif
Often found in proteins that regulate gene
transcription. A zinc is coordinated to
cysteine or histidine residues of the protein. An
a-helix is inserted into the major groove and
binds DNA.
histidine
histidine
zinc
Shown is azinc finger motiffrom a repressor
protein from a phage, a bacterial virus
cysteine
cysteine
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Zinc finger motif
Often found in proteins that regulate gene
transcription. A zinc is coordinated to
cysteine or histidine residues of the protein. An
a-helix is inserted into the major groove and
binds DNA.
cysteine
cysteine
Shown is azinc finger motiffrom the
glucocorticoid receptor, a protein thatmediates
hormone action. Two zincs are present.
zinc
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Leucine zipper motif
Often found in proteins that regulate gene
transcription. Two alpha helices interact
through interaction between hydrophobicleucine
amino acid side chains on one side of the alpha
helix.
Shown is aleucine zipper protein
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NextDetails of protein DNA interaction
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CAP
The catabolite activator protein(CAP) from E.
coli uses alphahelices to interact with
nucleotide bases in the majorgroove of the DNA
helix. This protein is an examplewhere a
helix-turn-helix motifis used to bind to
DNA. Binding of this protein to specific DNA
sequences preventsthe bacteria from utilizing
othersugars when glucose is present.
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Specific interactions of catabolite activator
protein (CAP) with DNA
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Specific interactions of catabolite activator
protein (CAP) with DNA
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Specific interactions of catabolite activator
protein (CAP) with DNA
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A closer look atprotein DNA interaction
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Specific interaction between proteins and
double-stranded B-DNA
Many DNA-binding proteins have an alpha helix
that interacts with nucleotide bases in the major
groove. An amine-containing amino acid side
chain often forms hydrogen bonds with major
groove bases.A single amino acid may form
hydrogen bonds with multiple, adjacent nucleotide
bases, increasing sequence-specific
interaction.Common amino acid arginine or
glutamine
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The DNA double helix contains a major groove and
a minor groove
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Binding to nucleotides in the major groove
The majority of the interactions between proteins
and DNA are hydrogen bonds with functional
groups in the major groove of the
double-stranded DNA molecule. Each DNA
binding protein recognizes specific sequences in
the DNA. Hydrogen bonding with N6 and N7 of
Adenine, O6 and N7 of Guanine,O4 of Thymine, and
N4 of Cytosine is possible.
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DNA binding proteins contain amino acids that
hydrogen bond to functional groups in the major
groove of DNA
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Regulatory Proteins
DNA sequences recognized by regulatory proteins
are often inverted repeats of a short DNA
sequence. These repeats form a palindrome with
two-fold symmetry about a central axis. DNA
binding proteins are often dimeric, with two
identical protein subunits. Each subunit
binds to one strand of the DNA.
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Binding of proteins to specific sequences in DNA
often controls the transcription of certain genes
Housekeeping genes genes for the enzymes of
central metabolic pathways. These genes are
expressed at a constant level. They are
constitutively expressed. Regulated genes genes
whose protein products rise and fall as cellular
demand increases or decreases. Expression can
be induced or repressed in the presence of
the appropriate metabolic signal. For example,
excess tryptophan in an E. coli cell will lead
to repression of the genes encoding enzymes of
tryptophan biosynthesis. The induction and
repression of transcription is mediated through
specific protein-DNA interactions.
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Most prokaryotic mRNA molecules are
polycistronic, they encode multiple genes
The genes are usually involved in the same
biochemical event A single promoter controls the
expression of these genes. This functional unit
of DNA is called an operon.
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Negative regulatory proteins bind to operator
sequencesin the DNA and prevent or weaken RNA
polymerase binding
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Positive regulatory proteins bind to the DNA and
enhance theinteraction of RNA polymerase with
the promoter region
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Specific protein DNA interactions
For a cell to function, proteins must distinguish
one nucleic acid sequence from another very
accurately. Activators and repressors of
transcriptionturn specific genes on and
off. Common themes of protein - DNA
interaction 1. Leucine zipper 2. Zinc finger 3.
Helix-turn-helix
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Zinc finger motif
Often found in proteins that regulate gene
transcription. A zinc is coordinated to
cysteine or histidine residues of the protein. An
a-helix is inserted into the major groove and
binds DNA.
cysteine
cysteine
Shown is azinc finger motiffrom the
glucocorticoid receptor, a protein thatmediates
hormone action. Two zincs are present.
zinc
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Another common DNA binding motif is a zinc
finger,a motif of 30 amino acids that
coordinates a Zn2 atom
The Zn2 atom is bound by eitherfour cysteines
or a combinationof two cysteines and two
histidines. A single protein can have multiple
zinc fingers,enhancing DNA binding.
Zn2
Zn2
Zn2
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The GATA transcription factor family contains
two zinc fingers for interaction with DNA
Cys-X2-Cys-X17-Cys-X2-Cys
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Steroid Hormones
Steroid hormones bind to soluble receptor
proteins in the nucleusof a cell and the
hormone-receptor complex binds to the
DNA,affecting transcription of steroid-activated
genes. Steroid hormonereceptors contain a
region of the protein that binds DNA usinga zinc
finger motif.
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Steroid Hormone Response Elements
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Interactions of the glucocortocoid receptor
protein with DNA
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Regulatory proteins that function as dimers
containregions of amino acid sequence that
mediate interactionbetween protein subunits
One common motif is the leucine zipper.
5-TACGGTACTGTGCTCGAGCACTGCTGTACT-3 3-ATGCCATGAC
ACGAGCTCGTGACGACATGA-5
central axis
Fig 28-14
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Leucine zipper motif
Often found in proteins that regulate gene
transcription. Two alpha helices interact
through interaction between hydrophobicleucine
amino acid side chains one one side of the alpha
helix.
The leucine residues of aleucine zipper
providehydrophobic interaction betweenalpha
helices at regular intervals. Every seventh
amino acidis a leucine.
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Helix-turn-helix motif
Often found in proteins that regulate gene
transcription. The pair of a-helices stack to
form a V shape with an angle of about 60º The
first helix positions the second helix. The
second helix binds to the DNA, projecting into
the major groove and recognizing specific
sequences.
An example of ahelix-turn-helix motifis found
in the cataboliteactivator protein (CAP)from E.
coli thatbinds to bacterial DNAand regulates
transcriptionof specific genes involvedin sugar
metabolism.
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Catabolite Activator Protein (CAP)
CAP (or CRP) binds to DNA in the presence of
cyclic AMP (cAMP). cAMP is high when glucose is
low and low when glucose is high. Transcription
of genes that metabolize lactose is activated
when thecAMP-CAP complexes binds to the DNA next
to the genes.
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Catabolite activator protein (CAP) bound to cAMP
and DNA
DNA
Region that interacts withRNA polymerase
cAMP
CRPmonomer 1
CRPmonomer 2
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Catabolite activator protein (CAP) bound to cAMP
and DNA
a-helix
The catabolite activator protein(CAP) from E.
coli uses alphahelices to interact with
nucleotide bases in the majorgroove of the DNA
helix. This protein is an examplewhere a
helix-turn-helix motifis used to bind to
DNA. Binding of this protein to specific DNA
sequences preventsthe bacteria from utilizing
othersugars when glucose is present.
a-helix
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Parkinson et al JMB 1996
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DNA sequence recognized by CAP
Underlined sequences are critical forprotein
DNA interaction
centralaxis
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Amino acid nucleotide interactions of
catabolite activator protein (CAP) with DNA
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CAP DNA complex (J. Mol. Biol. 1996. 260,
395-408)
Helix
Subunit 2
Subunit 1
Helix
Major groove
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CAP DNA complex
Helix-turn-helix
Subunit 2
Helix-turn-helix
Subunit 1
Major groove
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CAP DNA complex
DNAbindinghelix
Subunit 2
DNAbindinghelix
Subunit 1
Major groove
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CAP DNA complex
DNAbindinghelix
Subunit 2
DNAbindinghelix
Subunit 1
Major groove
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CAP DNA complex
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CAP DNA complex
CH3 of T
Arginine 180
N7 of G O6 of G
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CAP DNA complex
CH3 of T
Glutamate 181
CH3 of T
N4 of C
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CAP DNA complex
Arginine 185
CH3 of T
N7 of G O6 of G
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CAP DNA complex
Lysine 188
Phosphates
Serine 179
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CAP-DNA Contacts
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Parkinson et al JMB 1996
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Protein DNA interaction
Proteins often bind to specific sequences of
DNA.Example Restriction enzyme EcoRI binds to
the DNA sequence5-GAATTC-33-CTTAAG-5
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Restriction Fragment Length Polymorphism (RFLP)
A variation in sizes of DNA seen after cutting
with restriction enzymes. Restriction enzymes cut
DNA at a specific site. For example, the EcoR1
restriction enzyme cuts DNA whenever it sees the
letters GAATTC DNA before cutting by EcoR1
5-AATCTAGGGAATTCACAGCGATGCGAATTCGCAATTA-3 3-
TTAGATCCCTTAAGTGTCGCTACGCTTAAGCGTTAAT-5 DNA
after cutting by EcoR1 5-AATCTAGGG
AATTCACAGCGATGCG AATTCGCAATTA-3 3-TTAGATCCCTTA
A GTGTCGCTACGCTTAA GCGTTAAT-5 In this
example, EcoR1 has cut the one strand of 37 base
pairs into 3 smaller strands of DNA. If another
person has slightly different DNA, EcoR1 may cut
the DNA into pieces of different lengths. (For
example If the second GAATTC is GAATTT, EcoR1
will cut this other person's DNA in only one
place, producing 2 smaller strands of DNA.) The
words "fragment length polymorphism" mean "DNA
pieces of different lengths." RFLPs are a quick
way to see if two pieces of DNA are identical,
without having to look at the entire DNA sequence.
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Restriction Fragment Length Polymorphism (RFLP)
The RFLP analysis is a DNA fingerprinting method
that allows publichealth officials to
distinguish the transmission of specific strains
oftuberculosis during an outbreak. This method
is based on thedetection of the copy number and
location of the mobile geneticelement IS6110 in
the M. tuberculosis genome. Enzymaticdigestion
of the DNA produces fragments which can be
separatedby electrophoresis. The fragments are
immobilized on a nylonmembrane, and a specific
chemoluminescence-labeled DNA probe isused to
reveal the pattern bands on x-ray film. This
autoradiographof the banding is referred to as a
fingerprint http//www.cdc.gov/ncidod/dastlr/TB
/TB_RFLP.htm
Tuberculosis/Mycobacteriology BranchDivision of
AIDS, STD, and TB Laboratory ResearchNational
Center for Infectious DiseasesCenters for
Disease Control and Prevention
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IS6110 Fingerprints of M. tuberculosis
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Polymerase Chain Reaction-Restriction Fragment
Length Polymorphism Test For The Authentication
of Traditional Chinese Medicines
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DNA Profiling
Each person has a unique set of fingerprints. As
with a persons fingerprint no two individuals
share the same genetic makeup. This genetic
makeup, which is the hereditary blueprint
imparted to us by our parents, is stored in the
chemical deoxyribonucleic acid (DNA), the basic
molecule of life. Examination of DNA from
individuals, other than identical twins, has
shown that variations exist and that a specific
DNA pattern or profile could be associated with
an individual. These DNA profiles have
revolutionized criminal investigations and have
become powerful tools in the identification of
individuals in criminal and paternity cases.
The first widespread use of DNA tests
involved RFLP (restriction fragment length
polymorphism) analysis, a test designed to detect
variations in the DNA from different individuals.
In the RFLP method, DNA is isolated from a
biological specimen (e.g., blood, semen, vaginal
swabs) and cut by an enzyme into restriction
fragments. The DNA fragments are separated by
size into discrete bands in a gel (gel
electrophoresis), transferred onto a membrane,
and identified using probes (known DNA sequences
that are "tagged" with a chemical tracer). The
resulting DNA profile is visualized by exposing
the membrane to a piece of x-ray film which
allows the scientist to determine which specific
fragments the probe identified among the
thousands in a sample of human DNA. A "match" is
made when similar DNA profiles are observed
between an evidentiary sample and those from a
suspects DNA. A determination is then made as to
the probability that a person selected at random
from a given population would match the evidence
sample as well as the suspect. The entire
analysis may require from 6 to 10 weeks for
completion.
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restriction fragment length polymorphism ( RFLP)
Technique, also known as DNA fingerprinting, that
allows familial relationships to be established
by comparing the characteristic polymorphic
patterns that are obtained when certain regions
of genomic DNA are amplified (typically by PCR)
and cut with certain restriction enzymes. In
principle, an individual can be identified
unambiguously by RFLP (hence the use of RFLP in
forensic analysis of blood, hair or semen).
Similarly, if a polymorphism can be identified
close to the locus of a genetic defect, it
provides a valuable marker for tracing the
inheritance of the defect.
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Parentage Testing
The matching process for identifying DNA profile
patterns which either "exclude" or "include" a
person as being the parent of a child is shown in
the figure below. In this instance man 1 is
excluded from paternity and man 2 is included as
a possible father of the child.