4. By the end of this lecture you will be able to:
1. Identify the structural components of
deoxyribonucleic acid (DNA).
2. Define primary & secondary structure of
DNA[Watson and Crick model].
3. Differentiate between different forms of DNA.
4. Demonstrate the different levels of DNA
packaging
5. 1. Central Dogma of Molecular Biology.
2. Structure and function of nucleic acids.
3. Structural components of DNA: Bases,
Nucleosides and Nucleotides.
4. Primary structure of DNA.
5. Watson and Crick model of DNA
6. secondary structure.
7. Different forms of DNA structure.
8. Levels of DNA packaging
9. Nucleases and their specificity.
10. Denaturation of DNA and melting
temperature.
9. 1. REPLICATION (DNA SYNTHESIS)
2. TRANSCRIPTION (RNA SYNTHESIS)
3. TRANSLATION (PROTEIN SYNTHESIS)
THE FLOW OF GENETIC INFORMATION
(The Central Dogma of Molecular Biology)
10. DNA Structure
DNA organization & packing
Levels of DNA structure
Primary & Secondary structure
11. DNA Primary Structure
• DNA are formed of four nucleotides:
d-AMP, d-GMP, d-TMP and d-CMP.
• In each strand, nucleotides are linked
together by phosphodiester bonds
between the 3` hydroxyl of one nucleotide
and 5`-hydroxyl of the next nucleotide.
•The alternating sugar phosphate units
form the backbone of each DNA strand
(5`-P-S-P-S-P-3`).
•The nitrogenous bases, which are linked
to the pentoses, are projecting to the
inside of the two strands of DNA at right
angle.
•The sequence of bases determines the
coding structure of DNA (genetic
information).
16. DNA Primary Structure
• Each polynucleotide strand has two terminals.
• One end has a free phosphate group attached
to 5`-hydroxyl group of the terminal pentose
and the other end has a free 3`-hydroxyl
group.
• The order of nucleotides in any DNA strand is
always written in the 5` to 3` direction e.g.
AGCT for the following chain.
19. Exonucleases
Exonucleases cleave the last nucleotide
residue in either of the two terminals of an
oligonucleotide.
Endonucleases
Endonucleases cleave phosphodiester bonds
located in the interior of polynucleotides.
20.
21. DNA Secondary Structure
The B-form of DNA
• Watson and Crick
proposed a structure
for DNA in the form
of a double helix and
it is now referred as
B-form of DNA,
which is the most
common
physiological form.
22. 1- Two antiparallel strands :
• The two strands of DNA are paired to each other.
• The two strands run antiparallel, that is to say, one runs in
the 5`⇒ 3` direction and the other in the 3` ⇒ 5` direction.
Watson and Crick Model of DNA Structure
• The sugar/phosphate backbone
(hydrophilic) is on the outside
while the nitrogen bases
(hydrophobic) project into the
inside of the double helix.
23. The Two Chains of DNA Are Antiparallel
5’pCpGpApTpCpGpApT-OH3’
5’ pApTpCpGpApTpCpG-OH 3’
5’ 3’
3’ 5’
24. • The alternating sugar
phosphate units form the
backbone of each DNA
strand (5`-P-S-P-S-P-3`).
• The nitrogenous bases,
which are linked to the
pentoses, are projecting to
the inside of the two strands
of DNA at right angle.
25. Watson and Crick Model of DNA
Structure
2- Complementary base pairing:
• The two strands are held together by the
complementary base pairing through specific
hydrogen bonds.
- Adenine pairs with thymine through two
hydrogen bonds, and
- guanine pairs with cytosine through three
hydrogen bonds.
• Therefore the number of adenine bases equals
the number of thymine bases and the number of
guanine bases equals the number of cytosine
bases in DNA.
28. Watson and Crick Model of DNA
Structure
• The sequence of the two strands is complementary.
• The sequence of one strand determines the
sequence of the second one.
• This is important during DNA replication as each of
the original DNA strands acts as a template for
synthesis of a new complementary strand to form
two daughter DNA molecules.
29.
30. Watson and Crick Model of DNA Structure
3- Base stacking
• The base pairs inside the helix are stacked above each
other by Van der Waals forces and hydrophobic
interactions.
• The hydrogen bonding between complementary base
pairs and the Van der Waals forces and hydrophobic
interactions of stacked base pairs provide the stability of
the double helix.
31. 4- Spiral staircase:
The two strands coil around a common axis
to form a right-handed helix.
The double helix of DNA appears much like
spiral staircase, in which there is 10 base pairs
or steps for each complete turn of the helix.
32. Watson and Crick Model of DNA
Structure
5- Dimensions
• The B-form of DNA is 2 nm wide and each
complete turn is 3.4 nm long.
• From outside of the helix, two grooves are
apparent, a major groove (2.2 nm) and a minor
groove (1.2 nm).
• Through these grooves many drugs and proteins
can make contact with the nitrogenous bases
without any need to open the helix.
33.
34. Factors afftecting DNA double helix
stability
1. Hydrogen bonding : stabilize.
• Between complementary base pairs
• Relatively weak but additive and facilitates stacking.
2. Stacking interactions: stabilize.
• The Hydrophobic interactions & the Van der Waals
forces of stacked base pairs.
3. Electrostatic interactions: destabilize.
• Contributed mainly by negative charges of
phosphates
• Affect intrastrand and interstrand interactions.
• Repulsion can be neutralized with positive charges
(e.g., positively charged Na+ ions or proteins).
35. Comparison between Different Forms of DNA
A-Form B-Form Z-Form
One turn span Shorter Medium
(3.4 nm)
Longer
Diameter Thicker Medium Thinner
Number of bp /turn 11 10 12
Appearance of turn Smooth Smooth Zigzag
Direction of double
helix
Right Right Left
36. Denaturation of DNA
•Is the separation of the two
strands of DNA, due to rupture
of hydrogen bonds, and the
formation of single-stranded
DNA.
•Disruptions of the double-
stranded structure appear first in
regions of relatively high
adenine-thymine content.
•It occurs at:
• High temperatures.
• Extreme pH ranges or
• Extreme ionic strengths
37. Denaturation of DNA
• The size of these “bubbles”
increases with increasing
temperatures, leading to
extensive disruptions in the
structure of the double helix at
elevated temperatures.
• At higher temperatures the
double-stranded structure of
DNA is completely disrupted,
with the eventual separation of
the strands and the formation of
single-stranded open coils.
• Cooling of denatured DNA results
in reformation of the double helix or
renaturation.
38. • Melting Temperature (Tm):
• It is the temperature at which ½
of DNA helix is ruptured and
separated/
• DNA rich in A and T bases(2
hydrogen bonds) has lower Tm
than that rich in C and G bases
(3 hydrogen bonds).
43. ORGANIZATION OF
EUKARYOTIC DNA
• Human DNA that has a length of ~2m must be
condensed so that it can fit within a nucleus with a
diameter of ~10µm.
• In order to fit within nucleus, DNA should be made
compact by various types of sequential folding that
are stabilized by DNA binding proteins to form
chromatin.
– In non-dividing (interphase) cells:
• Chromatin is amorphous and dispersed throughout the
nucleus.
– Just prior to cell division (metaphase):
• Chromatin becomes organized into highly compacted
structures called chromosomes.
44. ORGANIZATION OF
EUKARYOTIC DNA
• Organization of eukaryotic DNA requires
2 classes of DNA-binding proteins:
• The histones
• The non-histone proteins
45. ORGANIZATION OF EUKARYOTIC DNA
• Eukaryotic DNA is associated with tightly bound
basic proteins called histones, which serve to order
the DNA into basic structural units called
nucleosomes that resemble beads on a string.
• Nucleosomes are further arranged into increasingly
more complex structures that serve to organize and
condense the long DNA molecules into
chromosomes that can be segregated during cell
division.
46. A. Histone proteins
• Histones are small proteins that are positively
charged at physiologic pH due to their high content
of lysine and arginine.
• There are five classes of histones, designated H1,
H2A, H2B, H3, and H4.
• Because of their positive charge, they form ionic
bonds with the negatively charged DNA.
• Histones, along with positively charged ions such as
Mg2+ help neutralize the large negative charge of the
DNA phosphate groups.
47. • They include:
the various transcription factors.
polymerases.
hormone receptors
other nuclear enzymes.
B- The non-histone proteins
48. Different levels of DNA “packing”
1. Nucleosomes (First level of packing):
•The nucleosome is formed of:
• Core of 8 histone molecules:
•Formed of 2 moleules of each
H2A, H2B, H3 & H4
• DNA:
– Around this core a segment of the DNA
double helix is wound nearly twice,
forming a negatively super-twisted helix.
– A linker DNA of about 50 base pairs
connect neighboring nucleosomes
• Role of H1 histone
– H1 binds to the linker DNA
– H1 facilitates the packing of
nucleosomes into the more compact
structures.
49. Nucleosome structure
Nucleosome core
146 bp DNA;
1 3/4 turns of DNA;
DNA is negatively supercoiled.
Two each: H2A, H2B, H3, H4
(histone octomer).
Nucleosome (chromatosome):
~200 bp DNA;
2 turns of DNA plus spacer;
Also includes H1 histone.
50. 2. Higher levels of organization:
• Polynucleosome, also called a nucleofilament, (Second level of
packing):
• Nucleosomes are packed more tightly to form a
polynucleosome (nucleofilament).
• This structure assumes the shape of a coil, often referred to
as a 30-nm fiber.
• The 30-nm fiber is organized into loops that are anchored to
nuclear scaffold proteins. (Third level of packing)
• Additional levels of organization lead to the final chromosomal
structure
51. Higher structure of DNA
Chromatin fibers are
organized into loops, and the
loops into the bands
that provide the superstructure
of chromosomes.
53. 1. First level of packing:
• It is by formation of nucleosomes.
• It produces 10 fold shortening of the length of DNA (11 nm in
diameter).
• Adjacent nucleosomes are connected by a short length of spacer
DNA giving rise to extended polynucleosome (nucleofilament).
2. Second level of packing:
• Nucleosomes can be packed more tightly to form a
polynucleosome involving 6 to 7 Nucleosome (chromatosomes)
per turn.
• This structure assumes the shape of a cylindrical coil (Solenoid).
• This leads to 50-fold shortening of the DNA (30 nm in diameter).
3. Third level of packing:
• The 30 nm fiber is organized into loops that are anchored by a
nuclear protein scaffold.
• Additional levels of organization lead to the final chromosomal
structure.
54. 1. Nucleosome core:
- Histone core + 1 ¾ turn (146 bp).
- Reduce DNA length by a factor of 10.
2. Nucleosome (Chromatosome) (11nm Diameter):
- Histone core + 2 turn (166 bp) + H1.
3. Nucleofilament (10 nm Diameter):
- Nucleosomes + Linker DNA ~20 – 90 bp.
- Extended polynucleosome chain.
- Has “ beads–on-a-string” appearance.
4. Polynucleosome (chromatin fiber) (30 nm Diameter):
- 6 to 7 nucleosomes per turn.
- Solenoid arrangment.
- Reduce DNA length by a factor of 50.
5. Metaphase chromosome (1400 nm Diameter).
* Histones may regulate DNA packaging by various in vivo reactions:
e.g. Methylation , acetylation & phosphorylation.
ORGANIZATION OF
EUKARYOTIC DNA
56. Fate of nucleosomes during DNA replication
• In order to replicate, the highly structured and constrained
chromatin must be relaxed.
• Dissociation of the nucleosome core from the DNA is
incomplete, (the parental histones remain loosely associated
with only one of the parental DNA strands).
• Synthesis of new histones occurs simultaneously with DNA
replication, and nucleosomes containing only newly
synthesized histones associate with only one of the new
daughter helices.
• Therefore, the parental histone octamers are conserved.
65. What is a genome?!
All the genetic
information
In eukaryotes it is
presented in
chromosomes
66. Genome structure
• Genome is the total genetic information
presented by the group of chromosomes
in any cell.
• The chromosomes that form the genome
differ in both length and number according
to the species.
68. • Viruses are composed of nucleic acids
enclosed in a protective protein coat
(capsid).
• The nucleic acids of viruses may be:
- A single or double stranded DNA (ssDNA
or ds DNA) OR
- A single or double stranded RNA (ssRNA
or dsRNA).
Viruses
71. Prokaryotic DNA and Chromosomes
• Prokaryotic organisms include bacteria
and blue- green algae.
Blue green algaeBacteria
72. Prokaryotic DNA and
Chromosomes
• Each cell contains one
single double-stranded
supercoiled circular
chromosome and has
no nuclear membrane.
• The chromosome is
associated with
histone-like proteins.
73. Prokaryotic DNA and Chromosomes
• Total chromosomal DNA codes for specific
proteins.
• The structural genes (nucleotide sequence
coding for proteins) do not always have
distinct physical locations on DNA.
• They frequently overlap with one another.
Protein A
Protein B
DNA
Gene B
Gene A
74. Prokaryotic DNA and
Chromosomes
• In addition, most species
of bacteria also contain
small and circular extra
chromosomal DNA
molecules called plasmids.
• Plasmid DNA carries
genetic information and
undergoes replication that
may or may not be
synchronized to
chromosomal division.
• Plasmids may carry genes
that convey antibiotic
resistance to the host
bacterium.
75. Eukaryotic DNA
• Only 2% of DNA code for proteins.
• The structural genes do not overlap.
• Eukaryotic genes are discontinuous (with few exceptions e.g. the
genes for histones & tRNA)
• Eukaryotic genes are formed of:
– Coding sequences:
• Called exons or expressed sequences
• They are unique and non-repetitive are interrupted by
– Non-coding sequences:
• Called introns or intervening sequences
• They are repetitive and
• Forms 25 – 35 % of the genome.
77. 1. Nuclear genome
• Human genome consists of:
• 46 (23 pairs) chromosomes
• With a total of 6×10 9 base pairs,
• Contains about 20,000 – 25,000 genes
2. Mitochondrial genome
• Circular genome of ~17,000 bp.
• Contains < 40 genes
Human Genome
78.
79. Genes:
• Vary in length from <100 to >2,300,000 bp.
• Most genes are single-copy in the haploid genome.
• Most of the eukaryotic genes are discontinuous
(with few exceptions e.g. the genes for histones &
tRNA) formed of:
• Exons (coding sequences which are unique and
non-repetitive) separated by
• Introns (non coding sequences which are
repetitive and forms 20 – 30 % of the genome
• Genes are composed of from 1 to >75 exons.
80. 5’ 3’
promoter
region
exons (filled and unfilled boxed regions)
introns (between exons)
transcribed region
translated region
mRNA structure
+1
Gene structure
81. The (exon- intron- exon)n structure of various genes
β-globin
HGPRTase
total = 1,660 bp; exons = 990 bp
Histone
Factor VIII
total = 400 bp; exon = 400 bp
total = 42,830 bp; exons = 1263 bp
total = ~186,000 bp; exons = ~9,000 bp
84. Test yourself
1. Which statement is true about the double helix:
a. Heating causes the strands to separate (denature).
b. GC pairs involve three hydrogen bonds.
c. Purine pairs with pyrimidine.
d. All of the above.
2. If a DNA molecule is composed of 40% (T) what percentage
of guanine would be expected:
a. 10%.
b. 20%.
c. 40%.
d. 80%.
3. All of the following are true about DNA EXCEPT:
a. Guanine usually pairs with cytosine and thymine with adenine.
b. A double helix formed of two antiparallel strands.
c. The sugar-phosphate backbone is positively charged.
d. Base stacking stabilizes the double helix.
85. Test yourself
4. Nucleases are enzymes that catalyze cleavage of:
a. Peptide bond.
b. Glycosidic bond.
c. Hydrogen bond.
d. Phosphodiester bond.
5. All of the following statements regarding the Watson-
Crick "B" form of DNA are true EXCEPT:
a. Two chains are coiled around a common axis forming
a right- handed helix.
b. The bases are found on the outside of the helix and the
sugar phosphate backbone on the inside.
c. The two chains run in opposite directions.
d. Adenine is always paired with thymine, guanine with
cytosine.
86. Test yourself
6. All of the following are true about eukaryotic genes EXCEPT:
a. Most of eukaryotic genes are discontineous.
b. They are always overlapping.
c. Coding sequences are unique and non repetitive.
d. They contain a regulatory sequence and a coding sequence.
7. The following statements describes both human and bacterial
DNA EXCEPT:
a. The DNA occurs physiologically as nucleosome complexes.
b. The DNA contains major and minor grooves.
c. The DNA consists of an antiparallel duplex.
d. The DNA contains equal molar fractions of adenine and thymine.
e. The DNA contains equal molar fractions of guanine and cytosine.
87. Test yourself
1. In DNA double helix, the alternating sugar
phosphate units form the backbone while
the nitrogenous bases are projecting to the
outside.
2. Exonucleases cleave phosphodiester bonds
located in the interior of polynucleotides.
3. The two strands of DNA double helix are held
together by the complementary base pairing
through specific hydrogen bonds.
4. Melting Temperature (Tm) is the temperature
at which the two strands of DNA double helix is
completely ruptured and separated.
5. Each DNA strand has two terminals one end has a
free phosphate group attached to 5`-hydroxyl group
of the terminal pentose and the other end has a free
3`-hydroxyl group.
88. Test yourself
6. Human immuno-deficiency virus (HIV) is RNA
virus; its genome is formed of two copies of dsRNA.
7. Prokaryotic genome consists of one single double-
stranded supercoiled circular chromosome
8. Plasmid DNA is small and circular extra chromosomal
DNA molecules that present in bacteria and
undergoes replication that is always synchronized to
chromosomal division.
9. Human genome consists of 46 chromosomes that
contain about 120,000 genes coding for about 120,000
proteins.
10. Most of the eukaryotic genes are continuous
sequences and genes are usually overlapping.
89. Test yourself
10. Most of the prokaryotic genes are continuous
sequences and genes are usually overlapping.
11. Eukaryotic genes consist of coding sequences
(exons) interrupted by intervening sequences (introns).
