Nucleic Acids

• Nucleic acid: are polymers of

Nucleotides linked with 3’, 5'
5’- phosphodiester bonds
• Nucleotide residues are all
oriented in the same 5' 3'
direction (5’ to 3’) giving the
polymer directionality.
• The sequence of DNA
3'
molecules is always read in
the 5’ to 3’ direction

On the 5’ end of the first nucleotide  Phosphate group

On the 3’ end of the first nucleotide & the 5’ end of the 2 nd nucleotide  Phosphate group so the
phosphodiester bonds are 3’ , 5’

The direction of reading is 5’  3’

 5'
5'

3'

Phospho diester bond 3' 3'

formation

5'

5'

3'

Esterification reaction between 2 nucleotides,

forming the linkage
Remember that the phosphodiester link is always 3’  5’

In the example above, we’ll read uracil guanine and NOT guanine uracil
 5'

3'

5'

3'

• Nucleotide monomers are joined by 3’-5’ phosphodiester linkages to

form nucleic acid (polynucleotide) polymers
•Phosphodiester bonds can be cleaved hydrolytically by
chemicals or enzymatically by deoxyribonucleases (DNA) or
ribonucleases (RNA)

5' 3'
 DNA
• 1o Structure - Linear array of nucleotides and their sequence
can be determined by different methods.
• 2o Structure – double helix
• 3o Structure - Super-coiling, stem-loop formation
• 4o Structure – Packaging into chromatin

DNA Secondary structure

• DNA is double stranded with antiparallel strands
• Right hand double helix
• Three different helical forms (A, B and Z DNA.

Primary structure  Simplest form , single strand.

 Properties of DNA Double Helix
* The two chains are coiled around a common axis
* The chains are paired in an anti-parallel manner
* Distance between the 2 sugar-phosphate
backbones is always the same, give DNA
molecule a regular shape.
* Plane of bases are oriented perpendicular to
backbone
* Hydrophilic sugar phosphate backbone winds
around outside of helix
* Noncovalent interactions between upper and
lower surfaces of base-pairs (stacking) forms
a closely packed hydrophobic interior.
* Hydrophobic environment makes H-bonding
between bases stronger (no competition with
water)

 Complementary is antiparallel.
 The backbone is sugar phosphate and it’s always fixed (The same)
 The sugar phosphate backbone is always hydrophilic, why?
Because the phosphate has a negative charge so it’s exists “"‫على األطراف‬
 The nitrogenous base is more on the inside and it’s complementary and antiparallel
Bases from two
adjacent DNA
strands can
hydrogen bond

•Adenine pairs with

thymine using two
H-bonds
•Guanine pairs with
cytosine using
three H-bonds

 The Adenine & Thymine and the Guanine & Cytosine form hydrogen bonds between them.
 Adenine & Thymine form 2 double bonds
 Guanine and cytosine form 3 double bonds (Stronger)
H-bonding of adjacent antiparallel DNA strands form
double helix structure

5'

3' 3'

5'

 After bonding to the complementary structure it forms double helix.

 Remember that we’re talking about 2nd structure and the primary structure is a single strand only.
 View down the Double Helix
Hydrophobic
Interior with base
pair stacking Sugar-phosphate
backbone

Pink  Sugar phosphate backbone

On the inside  Hydrophobic interior (Nitrogenous base).

 Structure of
DNA Double
Helix
• Right handed helix
• Rise = 0.33nm
nm/nucleotide
• Pitch = 3.4 nm /
turn
• 10.4 nucleotides
per turn
• Two groves – major
and minor

These measurements are fixed.

We’re not required to memorize these numbers but we need to know what they stand for.

Rise = Difference between 2 base pairs of the phosphate

Pitch = The whole turn

Major or minor are repetitive for each 2 turns, so we have a pattern

Ex : Minor, Major, Minor, Major, Minor etc..

 * Within groves, functional groups on
the edge of base pairs exposed to
exterior
* involved in interaction with proteins.
Factors stabilizing DNA double Helix
* Hydrophobic interactions – burying
hydrophobic purine and pyrimidine
rings in interior
* Stacking interactions – van der Waals
interactions between stacked bases.
* Hydrogen Bonding – H-bonding
between bases
* Charge-Charge Interactions –
Electrostatic repulsions of negatively
charged phosphate groups are
minimized by interaction with cations
(e.g. Mg2+)

When DNA starts forming secondary structures, it’ll form bonds to increase stability.

Within groves, functional groups on the edge of base pairs exposed to exterior which stabilizes the
double helix.

 Hydrophobic interaction is due to the plane (Rings aka purine and pyrimidine)
 We have repulsions due to phosphate’s negative charge .:. when the phosphate is wrapped around
itself, phosphate groups could get close to each other causing the repulsion.
 But to increase stability we minimize the negative charge repulsion due to cations.
Three major
structural forms
of DNA
A: right-handed,
short and broad,
2.3 A, 11 bp per
turn
B: right-handed,
longer, thinner,
3.32 A, 10 bp per
turn
Z: left-handed,
longest, thinnest,
3.8 A, 12 bp per
turn, Found in G:C-
rich regions of
DNA

 A & B  Right-handed , Z  Left-handed

 A  Short and broad (Less nucleotides)
 B  Longer and thinner (More nucleotides)
 Z  Longest and thinnest (Most nucleotides) . Rich in guanine and cytosine which looks like a Z
form
 The link between guanine and cytosine is the strongest, so it’s closer to each other
 Structural forms of the double helix
Three major structural forms of DNA
B-form, described by Watson and Crick
 It is right handed helix with 10 residues per 360ƒ turn of the helix
 The plane of bases perpendicular to helical axis
 Chromosomal DNA consists primarily of B-DNA
A-DNA form
 Is produced by moderately dehydrating the B form
 It is right-handed helix
 Contains 11 base pairs per turn
The planes of the base pairs are tilted 20 ƒ away from the
perpendicular to helical axis
Z-DNA form
 contains 12 bp per turn
Is left handed helix
Contains 12 about 12 base pair per turn
The deoxyribose phosphate backbone forms a “Zigzag structure”

 Chromosomal DNA is usually in the B (beta) form


Right handed helix
Structure of DNA Double Helix
Nucleoid
-Prokaryotic organism contains a single, double stranded,
supercoiled, circular chromosome, and it is associated with
histone-like proteins and some RNA to form a nucleoid
Plasmid
–Most species of bacteria contain small, circular,
extrachromosomal DNA molecules that called Plasmid.
–Plasmid DNA carries genetic information and undergoes
replication that may or may not related to the chromosomal
division.
–Plasmid carries genes that convey antibiotic resistance to
the host bacterium and may facilitate the transfer of
genetic information from one bacterium to another
–Plasmids used as vectors in recombinant DNA technology

Nucleoid  Prokaryotes.

Plasmid  Most bacteria.

Plasmid is extrachromosomal DNA (Extra, additional)

 Plasmid holds genetic information which gives the bacteria antibiotic resistance.
 It’s replicated
 is usually not related to the chromosome.
 Due to plasmid nature (it being something extra chromosomal as well as that it replicates in the
same rate as the bacteria) we benefited in synthesizing proteins such as insulin. (Recombinant
DNA technology)
 Separation of the two
DNA strands in the
double helix
Heating up to 70 – 90Cƒ the DNA
double helix denatures,
H-bonds are broken, bases
unstack, and the strands separate.

Renaturation (annealing) at lower

temperatures occurs in 2 steps.
1. Complementary bases pair.
2. The rest of the structure forms
cooperatively; it “zips-up”.
DNA with high G:C content
denatures at a higher Temp. than
A:T rich segments.

Replication of the DNA requires the double strand to break, but how is this going to happen?

It’ll happen by denaturation. If a part of the DNA is broken, it’s called “Partially denatured DNA”

If the double strand is connected again we call it “annealing”

And the denaturation, anneal is a cycle.

Denaturation  Heating

Annealing  Lowering temperature .

 DNA sequence Determines Melting Point

• Double Strand DNA can be

denatured by heat (get strand
separation)
• Can determine degree of
denturation by measuring
absorbance at 260 nm.
• Conjugated double bonds in
bases absorb light at 260 nm.
• Base stacking causes less
absorbance.
• Increased single strandedness
causes increase in absorbance

We can measure the melting point by measuring the absorbance on wavelength = 260nm.

Absorbance comes from the rings in DNA “Purine and pyrimidine”

Single strands usually have the highest absorbance possible, while double strands have lower absorbance.

Why?

Because when the bases are stacking (When adenine bonds to thymine for example) decreases absorbance

Focus on the peak

 Melting Point
Single strand has higher
relative absorbance at 260
than dose double stranded
DNA.
Increasing the
denaturation  increases
the absorbance
Melting point: the
temperature at which the
half of the helical
structure is lost

Melting point : the temperature which half of the DNA helix becomes single strand.
 DNA sequence Determines Melting
Point
* Melting temperature
related to G:C and
A:T content.
* 3 H-bonds of G:C
pair require higher
temperatures to
denture than 2 H-
bonds of A:T pair.

Remember than Guanine and Cytosine form 3 bonds & Adenine & Thymine form 2 bonds

So the guanine & cytosine will need higher melting point

The more the G&C in a DNA the higher the melting point.

The graph has 3 types of DNA

Pink  Equal ratio of A:T to C:G

 DNA 3o Structure
• Supercoiling
• Cruciform structures

DNA supercoiling:
• Supercoiling: means the coiling
of the coil.
• Typical phone cord is coiled like
a DNA helix and the coiled cord
can itself coil in a supercoil
• A number of measurable
properties of supercoiling have
been established

Tertiary structure is somewhat similar to proteins

After a secondary structure is formed, we do supercoiling to get tertiary structure.

 Significance of DNA Supercoiling:

E.Coli is an example on supercoiling

In DNA there are 2 strands that are wrapped around each other

After they’re wrapped they make a circular structure then makes another circle (Which is what we call
supercoiling)
 DNA Supercoiling

•Supercoiling is a property of
circular DNA not linear
•The number of helical turns
in a linear 260-bp DNA duplex
in the B-DNA form is 25
(260/10.4)
•Joining the two ends
Relaxed circular DNA (also has
25 helical turn)

The circular form is the form that’s capable of doing supercoiling.

Circular form is also a property of DNA

 Another circular DNA can be formed by unwinding the linear duplex
by two turns before joining its ends

This circular DNA

contains 23 turns of B-
helix and unwound loop

In vivo most DNA is negatively supercoiled

Circular DNA can also do unwinding which is mandatory for replication.

 A supercoiled DNA molecule is more compact than a relaxed DNA
molecule of the same length supercoiled DNA moves faster than
relaxed DNA in centrifuged
Topological parameters that describe supercoling
Linking number Lk: the number of times one strand of DNA winds
around the other in the right-handed direction. Molecules that differs
only in linking number are called topological isomers or topoisomers
 Number of turns of Watson-Crick helix “T” (Twisting number)
 Number of turns o superhelix “W” (writhing number)
L=T + W
T, W can be non-integral but L should be integral
Supercoiled DNA is favored over unwound DNA because it contains
more paired bases
Enzymes called topoisomerases or gyrases can introduce or remove
supercoils

Supercoiled is more compact

Topoisomers : differ in which side did the strand wind (Right or left handed)

Supercoiling is favorable because its more compact and takes less size.
 DNA Supercoiling

Positive = Clockwise

Negative = Counter Clockwise

 Certain DNA sequences adopt unusual structures
•Palindrome: a word that spelled identically reading forward or backward
(ROTATOR).
•This term is applied to DNA with inverted repeats of base sequence having
twofold symmetry over two strands of DNA, such sequences are self-
complementary within each strand
• When inverted repeat occurs within each individual strand of the DNA, the
sequence is called a mirror repeat.

Palindrome = ‫ خوخ‬, ‫ توت‬etc

Remember that palindromes are for TWO strands.

Palindromes are self-complementary.

Hairpin is another structure that is a property of tertiary structure.

Cruciform = They do a cross

What does that mean?

Each strand does its own hairpin.

Mirror repeat only happens on 1 strand CACCAC

They can’t do hairpin or cruciform

They are not self-complementary

 Palindromes have potential to form hairpin or cruciform

tRNA and DNA have hairpin

Cruciform: cross-shaped
structure
Can form intrachain base
pairing
 DNA 4o Structure: Chromosome Structure
• In chromosomes, DNA is tightly associated with proteins
• Human DNA’s total length is ~2 meters!
• This must be packaged into a nucleus that is about 5 micrometers in
diameter
• This represents a compression of more than 100,000!
• It is made possible by wrapping the DNA around protein spools called
nucleosomes and then packing these in helical filaments
Nucleosome Structure
• Chromatin, the nucleoprotein complex, consists of histones and
nonhistone chromosomal proteins
• % major histone proteins: H1, H2A, H2B, H3 and H4
• Histone octamers are major part of the “protein spools”
• Nonhistone proteins are regulators of gene expression

Quaternary is associated with protein which forms “nucleosome”

Quaternary structure is also called chromosome structure

In DNA we have secondary structure which has a tertiary structure wrapped then we have proteins
“nucleosome” which form a complex called nucleoprotein

Nucleoproteins : Normal proteins that are inside a nucleus which form chromatin

Nucleoproteins are made out of histones and non-histone chromosomal protein.

Histone protein form octamers.

Histones are nucleoproteins, are usually made out of amino acids.

Most amino acids that form histones are positive amino acids such as lysine and arginine

Why?

Because the DNA backbone is phosphate rich (which is negative) so to avoid repulsion.
 Nucleosome Structure
• High content of Lysine and
arginine (+ve charge)
•4 major histone (H2A, H2B,
H3, H4) proteins for octomer
•200 base pair long DNA
strand winds around the
octomer
•146 base pair DNA “spacer
separates individual
nucleosomes
•H1 protein involved in higher-
order chromatin structure.
•Chromatin looks like beads on
string

Nucleosome works as a small nucleus that DNA is wrapped around, which decreases the length of a DNA

We have hundreds of nucleosomes that are wrapped and become “closer” to each other which makes it
easier to pack DNA.
Organization of
Eukaryotic DNA
Organization of Eukaryotic DNA
Slide 36

The End
Slide 37
•4 major histone (H2A,
H2B, H3, H4) proteins for
octomer
•200 base pair long DNA
strand winds around the
octomer
•146 base pair DNA “spacer
separates individual
nucleosomes
•H1 protein involved in
higher-order chromatin
structure.
• W/O H1, Chromatin looks
like beads on string