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DNA structure- Part II
Levels of DNA structure
23/05/2017
2-1
Levels of DNA structure
 1°structure: the order of
bases on the polynucleotide
sequence; the order of bases
specifies the genetic code.
 2°structure:
the
threedimensional conformation of
the polynucleotide backbone.
 3°structure: supercoiling.
 4°structure:
interaction
between DNA and proteins
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DNA - 1° Structure
• Deoxyribonucleic
acids
(DNA) is a biopolymer that
consists of a backbone of
alternating units of 2-deoxyD-ribose and phosphate.
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Continue….
This
bond
which
indicates that there are
two covalent bonds
formed between the OH and the acidic
phosphate group
The 3’-OH of one 2deoxy-D-ribose
is
joined to the 5’-OH of
the next 2-deoxy-Dribose with bases by a
phosphodiester bond.
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Continue….
So, the Primary Structure is
the sequence of bases along
the
pentose-phosphodiester
backbone of a DNA molecule
 Base sequence is read from
the 5’ end to the 3’ end.
System of notation single
letter (A,G,C,U and T).
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 The backbone of DNA & RNA is hydrophilic.
 The hydroxyl gp (OH) of the sugar residues form hydrogen bonds with
water.
 The Phosphate gp, with pka  2, are completely ionized and –vely
charged at pH 7.
 The –ve charges are neutralized by ionic interactions with +ve charges
on proteins, metal ions.
 A short nucleic acid containing 50 or fewer nucleotides is called an
oligonucleotide, a longer is a polynucleotide.
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the 2’
hydroxyl
is
absent in
DNA
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Abbreviations of
Nucleic Acid Sequences
• pA-C-G-T-AOH
• or pApCpGpTpA
• or pACGTA
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Nucleotide Bases affect the three
dimensional structure of nucleic acids
• The purine and pyrmidine
bases are conjugated ring
system.
• One result is that pyrmidines
are planar molecules and
purines are nearly planar with
a slight pucker.
• Free purine and pyrimidine
bases can exist in two or more
forms
called
tautomers,
depending on pH.
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A tautomeric shift occurs when a
proton
changes
its
position,
resulting in a rare tautomeric form.
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Continue….
•
Standard and anomalous basepairing arrangements that occur if
bases are in the rare tautomeric
forms. Base mispairings due to
tautomeric shifts were originally
thought to be a major source of
errors in replication.
• Such structures have not been
detected in DNA, and most evidence
now suggests that other types of
anomalous pairings are responsible
for replication errors.
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Continue….
• As
a result of resonance,
delocalized electrons in the
conjugated ring rings are
available to absorb UV light at
260nm.
• The chemical properties of the
purine and pyrimidines give rise
to two modes of interaction
between bases:
As a result of resonance, all
nucleotide bases absorb UV light.
1. Hydrophobic stacking, the bases
relatively insoluble in water at
neutral pH.
2. Base pairing which result from Hbonding .
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Nucleotides play additional roles in cells
•
Adenosine is a building block
for some important enzyme
cofactor, such as NAD+ and FAD.
The presence of an adenosine
component in a variety of
cofactors enables recognition by
enzymes that share common
structural features.
NAD+
FAD
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Continue….
• cAMP formed from
ATP in a reaction
catalyzed by adenylyl
cyclase, is a common
second
messenger
produced in response
to hormones and other
chemical signals
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DNA - 2° Structure
• Secondary structure is the ordered
arrangement of nucleic acid strands.
• Double helix is a type of 2° structure of
DNA molecules in which two antiparallel polynucleotide strands are
coiled in a right-handed manner about
the same axis.
•
Structure
based
crystallography.
on
X-Ray
• The molecule is stabilized by
hydrophobic interactions in its
interior and by hydrogen bonding
between the complementary bases
pairs A-T and G-C
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Base Pairing
• Base
pairing
is
complementary.
• A major factor stabilizing the
double helix is base pairing by
hydrogen bonding between TA and between C-G
 T-A base pair comprised of 2
hydrogen bonds.
 G-C base pair comprised of 3
hydrogen bonds
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Base stacking
 Bases are hydrophobic and interact by van der
Waals interactions.
 In standard B-DNA, each base rotated by 32°
compared to the next and, while this is perfect for
maximum base pairing, it is not optimal for
maximum overlap of bases; in addition, bases
exposed to the minor groove come in contact with
water.
 Many bases adopt a propeller-twist in which base
pairing distances are less optimal but base stacking
is more optimal and water is eliminated from
minor groove contacts.
 The propeller twist of the base pairs results in
purine-purine clash in the center of the helix.
Because the purines are larger than the pyrimidine
rings, they extend beyond the helical axis of DNA.
Hydrophobic, van der Waals, and
electrostatic interactions favor the
alignment of bases in an aqueous
solution or within a polynucleotide
chain the un-stacked orientation is
disfavored.
DNA attempts to reduce purine-purine clash
in several ways:
A. The base pairs rotate less along the helix axis in the purine-pyrimidine
sequences (lower average helical twist).
They tend to rotate less in the pyrimidine-purine sequences (lower
than average helical twist).
The average helical twist was still very close to the 36° proposed by
Watson and Crick.
B. Another way DNA minimizes the purine-purine clash is that it bends
toward the minor grove or major groove to reduce the interaction.
C.
Finally clashing base pairs could slide left or right toward the
phosphodiester backbones to minimize the purine-purine interaction.
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Major and minor grooves
 The major groove is large
enough to accommodate an
alpha helix from a protein.
 Regulatory
proteins
(transcription factors) can
recognize the pattern of
bases
and
H-bonding
possibilities in the major
groove.
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DNA adopts different helical forms
• Nucleic acids are inherently flexible molecules. Numerous bonds
in the sugar-phosphate backbone can rotate, and thermal
fluctuation can lead to bending, stretching, and un-pairing of the
two strands.
•
As a result, cellular DNA contains significant deviations from
the Watson-Crick DNA structure, some or all of which may play
important roles in DNA metabolism.
• Generally, such structural variations do not affect the key
properties of strand complementarity: antiparallel strands and
the requirement for AT and G≡C base pairs.
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Variation in the three-dimensional structure of DNA
reflect three things:
(1) The different possible
confirmations of the deoxyribose.
(2) Rotation about the contiguous bonds that
make up the sugar-phosphate backbone.
(3) Free rotation about the glycosidic bond.
Continue….

B-DNA (Watson-Crick Form)
 considered the physiological form
 a right-handed helix, diameter 11Å
 10 base pairs per turn (34Å) of the helix.

A-DNA (favored in environment with very
low water content)
 a right-handed helix, but thicker than
B-DNA.
 11 base pairs per turn of the helix
has not been found in vivo.

Z-DNA occurs at high salt concentrations in
polymers that have a sequence of alternating
purines and pyrimidines
 a left-handed double helix.
 may play a role in gene expression.
 Z-DNA occurs in nature, usually consists of
alternating purine-pyrimidine bases.
 Methylated cytosine found also in Z-DNA.
In each case, the sugar-phosphate backbones wind around the exterior of the helix (red and
blue), with the bases pointing inward. The same 25-base-pair DNA sequence is shown in all three
forms. Differences in helical diameter can be seen in end-on views (top); differences in helical
rise and groove shape are apparent in the side views (bottom). B-DNA, the most common form in
cells, has a wide major groove and a narrow minor groove. A-form helices, common for RNA and
certain DNA structures, are more compact than B-DNA. The major groove is deeper and the
minor groove is shallower than in B-DNA. Z-DNA, which forms only under high salt conditions or
with C≡G-rich DNA sequences, is left-handed, and its backbone has a zigzag pattern. It is less
compact than B-DNA, with a very shallow major groove and a narrow and deep minor groove.
DNA - 3° Structure
•
Tertiary structure is the three-dimensional
arrangement of all atoms of a nucleic acid;
commonly referred to as supercoiling.
•
The term "supercoiling" means literally the
coiling of a coil.
•
DNA is coiled in the form of a double helix.
•
Enzymes called topoisomerases or gyrases
can introduce or remove supercoils.
If there is no net bending of the DNA axis
upon itself, the DNA is said to be in
a relaxed state.
Negative supercoiling may promote
cruciforms
•
•
Certain DNA Sequences Adopt Unusual Structures
• Other sequence-specific DNA structures have been detected,
within larger chromosomes, that may affect the function and
metabolism of the DNA segments in their immediate vicinity.
• For example, certain repetitive sequences can bend the DNA
helix in a distinct way.
•
This DNA bending helps certain proteins—such as
transcription factors, which promote the synthesis of
mRNAs—bind to their target DNA binding sites.
•
Regions of DNA where the two complementary strands have
the same sequence when read in the 5′→3′ or the 3′→5′
direction occur relatively frequently in chromosomal DNA and
are called palindromes.
Continue….
•
•
•
•
•
In language, a palindrome is a word, phrase, or sentence that is spelled
identically when read either forward or backward.
Two examples are ROTATOR and NURSES RUN.
In biology, the term applies to double-stranded regions of DNA where one
strand’s sequence is identical to its complement.
for example, 5′-GAATTC-3′ is a palindrome because its complementary
sequence is also 5′-GAATTC-3′.
Palindromes are formed from adjacent inverted repeats, which can occur
within one strand of DNA or over the two strands of the double helix
Continue….
• These sequences play important biological roles, such as:
 Slowing or blocking protein synthesis by the ribosome-a process
called translation attenuation .
 Forming recognition sites for restriction enzymes, which
catalyze double-stranded DNA cleavage.
DNA classes and sizes
Circular DNA is a type of
double-stranded DNA in
which the 5’ and 3’ ends of
each stand are joined by
phosphodiester bonds.
Human chromosome DNA
Plasmid DNA
E. Coli DNA
λ phage DNA
Gene
What is in the
middle??????
Protein
RNA Structure
• In the early 1970s, Alexander
Rich, Aaron Klug, and Sung-Hou
Kim independently solved the
structures of transfer RNAs,
revealing how tRNAs carry the
amino acids that are used in
protein
synthesis
on
the
ribosomes.
• The wide-ranging functions of
RNA reflect a structural diversity
much richer than that observed in
DNA molecules.
Continue….
• The single strand of RNA folds back on itself to form
short base-paired or partially base-paired segments
connected by unpaired regions.
• This property, called RNA secondary structure, enables
RNA molecules to fold into many different shapes that
lend themselves to many different biological functions.
Continue….
• The greater structural variety in RNA relative to DNA reflects the three
main chemical differences between the two polynucleotides:
1- The pentose (2′-deoxyribose in DNA vs. ribose in RNA).
2- The base composition (thymidine vs. uridine).
3- The sugar pucker of the pentose (C-2′ endo vs. C-3′ endo).
•
Double-stranded RNAs do exist in nature, such as those that form the
genomes of some viruses.
• In addition, some RNAs do not seem to form stable three-dimensional
structures from local base-pairing interactions, e.g. mRNA.
• These RNAs may fold into three-dimensional structures only in the
presence of bound proteins, forming complexes called
ribonucleoproteins (RNPs).
RNAs Form Various Stable Three-Dimensional
Structures
• Most of the highly structured RNAs contain noncanonical base
pairs and backbone conformations not observed in DNA.
• In many cases, the 2′-hydroxyl group on ribose, a chemical feature
that distinguishes RNA from DNA, seems to be directly or
indirectly responsible for these unique structural properties.
• The presence of the 2′ hydroxyl makes RNA vulnerable to
hydrolysis, but it also allows for additional hydrogen bonding
between segments of an RNA molecule.
•
As a result, RNA helices are more thermodynamically stable than
are DNA helices of the same length and sequence.
Continue….
• Base pairs other than canonical AU and CG pairs are common in RNAs,
including A-A and G-U. In all cases, base pairs or single bases are most
stable when stacked on top of one another in a helix.
• Divalent and monovalent metal ions (Mg2+, Ca2+, K+, and Na+) bind to
specific sites in RNA and help shield the negative charge of the backbone,
allowing parts of the molecule to pack more tightly together.
RNA is very similar to DNA
Chemically, RNA is very similar to DNA. There are some main differences:
1- RNA uses the sugar ribose instead of deoxyribose in its backbone.
Vicinal Hydroxyl Group Makes Difference !!
 The vicinal OH groups of RNA are
susceptible to nucleophilic attach leading to
hydrolysis of the phosphodiester bond.
 DNA is not susceptible to
hydrolysis. RNA is alkali labile.
alkaline
Continue…
2- RNA uses the base Uracil (U) instead of Thymine (T).
U is also complementary to A.
But Why DNA Contains Thymine rather than Uracil?!
 It is because cytosine deaminates to form
uracil in a finite rate in vivo. This would
results in a mutation in the DNA:
 So any U found in DNA will be corrected
by a “proofreads” system. Thus DNA can
not normally have U.
Guanine and cytosine form a base pair stabilised by three hydrogen bonds, whereas
adenine and thymine bind to each other through two hydrogen bonds. The red frames
highlight the functional groups of cytosine and thymine that are responsible for
forming the hydrogen bonds. Cytosine can spontaneously undergo hydrolytic
deamination, resulting in a uracil base with the same capability for hydrogen bond
formation as thymine.
Continue…
3- RNA tends to be single-stranded.
4- Functional differences between RNA and
DNA.
 DNA single function, RNA many functions
according to their type.
 Example of types of RNA e.g: tRNA, mRNA,
rRNA.
The Central Dogma
 The Flow of Information: DNA → RNA → Protein.
 A gene is expressed in two steps:
• DNA is transcribed to RNA.
• Then RNA is translated into protein.
Many RNA molecules do not encode proteins
tRNA.
rRNA.
a vast number of other non-coding RNAs
(ncRNAs).
How big part of human transcribed RNA
results in proteins?
 Of all RNA, transcribed in higher eukaryotes, 98%
are never translated into proteins.
 Of those 98%, about 50-70% are introns.
 4% of total RNA is made of coding RNA.
 The rest originate from non-protein genes,
including rRNA, tRNA and a vast number of other
non-coding RNAs (ncRNAs).
 Even introns have been shown to contain ncRNAs,
for example snRNAs.
 It is thought that there might be order of 10,000
different ncRNAs in mammalian genome
The “Other” 98% of the Human
Genome
ncRNA genes have diverse and essential
roles.
May be relics of ancient RNA-based life.
Many cellular machines contain RNA.
• Ribosome rRNA
•
•
Spliceosome snRNAs (U1,U2,U4,U5,U6)
Telomerase Telomerase RNA
RNA types
Non-coding RNA. Transcribed
RNA with a structural,
functional or catalytic role
mRNA
rRNA Participate
in protein
synthesis
tRNA Interface
between mRNA &
amino acids
snRNA: RNA that
form part of the
spliceosome
stRNA- Small temporal
RNA, with a role in
developmental timing
snRNA found in
nucleolus, involved in
modification of rRNA.
miRNA involved
regulation of
expression
Other large RNA
with roles in
chromotin structure
siRNA- Small interfering
RNA, Active molecules in
RNA interference
RNA molecules are classified according to their structure
and function
The Role of Different Kinds of RNA
RNA Type
Size
Function
tRNA
Small
Transport amino acids to site of protein synthesis
rRNA
Variable size
Combines with proteins to form ribosomes, the site
of protein synthesis
mRNA
Variable
Direct amino acid sequence of proteins.
snRNA
Small
mRNA to it is mature form in eukaryotes
Processes initial
siRNA
Small
Affect gene expression
miRNA
Small
Affect gene expression
tRNA
 The smallest kind of the three RNAs.
 A single-stranded polynucleotide chain between 73-94
nucleotide residues.
 Intramolecular hydrogen bonding occurs in tRNA.
tRNA is a cloverleaf shaped, single strand.
It has:
• Anticodon loop.
• Amino acid binding site at the 3’end.
• Two other loops for binding the ribosomes.
rRNA




rRNA: a ribonucleic acid found in ribosomes, the site of protein synthesis.
Only a few types of rRNA exist in cells.
Ribosomes consist of 60 to 65% rRNA and 35 to 40% protein.
In both prokaryotes and eukaryotes, ribosomes consist of two subunits,
one larger than the other.
mRNA
 A ribonucleic acid that carries coded genetic information from DNA
to ribosomes for the synthesis of proteins.
 Present in cells in relatively small amounts and has a short-half life.
 Single stranded and unstable.
 Biosynthesis is directed by information encoded on DNA.
 A complementary strand of mRNA is synthesized along one strand
of an unwound DNA, starting from the 3’ end.
Eukaryotic mRNA Structure


Capping: linkage of 7-methylguanosine 7 to the 5’ terminal
residue.
Tailing: attachment of an adenylate polymer (poly A)
Chemical and thermodynamic importance
of the DNA structure
Denaturation and renaturation
Complementary base paring by hydrogen bonding and van der
Waals interaction storage and transfer of genetic information
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Properties of DNA- Denaturation
 When DNA is heated to 80OC, its UV absorbance increases by 30-40% .
 With heating, noncovalent forces holding DNA strands together weaken and
break.
 When the forces break, the two strands come apart in denaturation or
melting.
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Denaturation of DNA
 As strands separate, absorbance at 260 nm increases.
 Stacked base pairs in native DNA absorb less light .
 Increase is called hyperchromicity.
 Temperature at which DNA strands are ½ denatured is
the melting temperature or Tm.
 Melting Temperature (Tm) – the temp. at which half of
the helical structure is lost
Hyperchromic Effect – a large increase in light absorbance at 260
nm occurring as double-helical DNA is melted (i.e. unwound).
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Melting temperature and % G-C
 GC content of DNA has a significant
effect on Tm with higher GC content
meaning higher Tm.
Examples
 Upon denaturation, the Hbonds between the base pairs
of a native nucleic acid are
replaced by energetically
equivalent hydrogen bonds
between the bases and water
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Continue…
• In addition to heat, DNA
can be denatured by:
 Organic solvents.
 High pH.
 Low salt concentration.
• GC content also affects
DNA density
 Direct, linear relationship
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DNA Renaturation
• After two DNA strands separate, under proper
conditions the strands can come back together
• Process is called annealing or renaturation
• Three most important factors:
 Temperature – best at about 25 C below Tm.
 DNA Concentration – within limits higher concentration
better likelihood that 2 complementary will find each
other.
 Renaturation Time – as increase time, more annealing
will occur
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Summary
• GC content of a natural DNA can vary from less
than 25% to almost 75%.
• GC content has a strong effect on physical
properties that increase linearly with GC content.
 Melting temperature, the temperature at which the two
strands are half-dissociated or denatured.
 Density.
 Low ionic strength, high pH and organic solvents also
promote DNA denaturation.
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Hybridization
Annealing of complementary DNA (hybrid duplex) from different
species at 65℃.
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Continue…
• Hybridization is a process of
putting together a combination of
two different nucleic acids.
 Strands could be 1 DNA and 1 RNA.
 Also could be 2 DNA with
complementary
or
nearly
complementary sequences.
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DNA Sizes
• DNA size is expressed in 3
different ways:
 Number of base pairs .
 Molecular weight – 660 is
molecular weight of 1 base
pair.
 Length – 33.2 Å per helical
turn of 10.4 base pairs.
• Measure DNA size either using
electron microscopy or gel
electrophoresis.
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Summary
• Natural DNAs come in sizes ranging from several
kilobases to thousands of megabases.
• The size of a small DNA can be estimated by
electron microscopy.
• This technique can also reveal whether a DNA is
circular or linear and whether it is supercoiled.
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