Nature of Genetic

Material

IDENTIFICATION OF DNA AS
THE GENETIC MATERIAL
To fulfill its role, the genetic material must meet several
criteria:
Information: It must contain the information necessary to. 1
make an entire organism
Transmission: It must be passed from parent to offspring. 2
Replication: It must be copied. 3
In order to be passed from parent to offspring

Variation: It must be capable of changes. 4

To account for the known phenotypic variation in each species

Frederick Griffith Experiments

with Streptococcus pneumoniae
Griffith studied a bacterium (Diplococcus pneumoniae)
now known as Streptococcus pneumoniae
S. pneumoniae comes in two strains
S Smooth
Secrete a polysaccharide capsule
Protects bacterium from the immune system of animals
Produce smooth colonies on solid media
Virulent

R Rough
Unable to secrete a capsule
Produce colonies with a rough appearance
Non Virulent

 In 1928, Griffith conducted experiments using two

strains of S. pneumoniae: type IIIS and type IIR
1. Inject mouse with live type S bacteria
Mouse died
Type S bacteria recovered from the mouses blood

2. Inject mouse with live type R bacteria

Mouse survived
No living bacteria isolated from the mouses blood

3. Inject mouse with heat-killed type S bacteria

Mouse survived
No living bacteria isolated from the mouses blood

4. Inject mouse with live type R + heat-killed type S cells

Mouse died
Type S bacteria recovered from the mouses blood

 Griffith concluded that something from the dead

type S transfored type R into type IIIS
He called this process transformation

The substance that allowed this to happen was

termed the transformation principle
Griffith did not know what it was

The nature of the transforming principle was

determined using experimental approaches that
incorporated various biochemical techniques
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9-9

Hershey and Chase Experiment

with Bacteriophage T2
In 1952, Alfred Hershey and Marsha Chase
provided further evidence that DNA is the genetic
material
Figure 9.4

They studied the

abacteriophage T2
It is relatively simple
since its composed of
only two
macromolecules

Inside the
capsid

Made up
of protein

DNA and protein

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-12

Figure 9.5
Life cycle of the
T2
bacteriophage

The Hershey-Chase
Experiment

Viruses that infect

bacteria have a simple
structure
DNA core surrounded
by a protein coat

Hershey and Chase used

two different radioactive
isotopes to label the protein
and DNA
Incubation of the labeled
viruses with host bacteria
revealed that only the DNA
entered the cell
Therefore, DNA is the
genetic material

Thus, viral DNA

directs the
production of new
viruses

The Hershey and Chase experiment can be

:summarized as such
Used radioisotopes to distinguish DNA from proteins
32P labels DNA specifically
35S labels protein specifically
Radioactively-labeled phages were used to infect
nonradioactive Escherichia coli cells
After allowing sufficient time for infection to proceed,
the residual phage particles were sheared off the cells
Phage ghosts and E. coli cells were separated=<
Radioactivity was monitored using a scintillation
counter

Only the genetic material of the phage is injected

into the bacterium
Isotope labeling will reveal if it is DNA or protein

NUCLEIC ACID STRUCTURE

DNA and RNA are large macromolecules with
several levels of complexity
Nucleotides form the repeating units. 1
Nucleotides are linked to form a strand. 2
Two strands can interact to form a double helix. 3
The double helix folds, bends and interacts with. 4
proteins resulting in 3-D structures in the form of
chromosomes

Three-dimensional structure

Nucleotides
The nucleotide is the repeating structural unit of
DNA and RNA
It has three components
A phosphate group
A pentose sugar
A nitrogenous base

These atoms are found within individual nucleotides

However, they are removed when nucleotides join together to make

strands of DNA or RNA
A, G, C or T

A, G, C or U

The structure of nucleotides found in (a) DNA and (b) RNA

 Base + sugar nucleoside

Example
Adenine + ribose = Adenosine
Adenine + deoxyribose = Deoxyadenosine

Base + sugar + phosphate(s) nucleotide

Example
Adenosine monophosphate (AMP)
Adenosine diphosphate (ADP)
Adenosine triphosphate (ATP)

Base always
attached here

Phosphates are
attached there

 Nucleotides are covalently linked together by

phosphodiester bonds
A phosphate connects the 5 carbon of one nucleotide to
the 3 carbon of another

Therefore the strand has directionality

5 to 3

The phosphates and sugar molecules form the

backbone of the nucleic acid strand
The bases project from the backbone

O
Base
P O CH2
O
O 5
4
1
H
H
H
H
2
Phosphate 3
O
H
2 Deoxyribose
Repeating unit of
deoxyribonucleic
acid (DNA)

O
Base
P O CH2
O
O 5
4
1
H
H
H
H
Phosphate 3
2
O
OH
Ribose
Repeating unit of
ribonucleic acid (RNA)

A Few Key Events Led to the

Discovery of the Structure of DNA
In 1953, James Watson and Francis Crick
discovered the double helical structure of DNA
The scientific framework for their breakthrough was
provided by other scientists including
Linus Pauling
Rosalind Franklin and Maurice Wilkins
Erwin Chargaff

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9-31

Linus Pauling
In the early 1950s, he
proposed that regions of
protein can fold into a
secondary structure
-helix

To elucidate this structure,

he built ball-and-stick
models
Refer to Figure 9.12b

Figure 9.12

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-32

Fig. 9.13

Rosalind Franklin

Photo 51 x-ray diffraction of wet DNA

showing the B form double helix
taken by Rosalind Franklin and Raymond Gosling on Friday, 2nd May, 1952
by long exposure started the previous day

Rosalind Franklin
She made marked advances in X-ray diffraction
techniques with DNA
The diffraction pattern she obtained suggested
several structural features of DNA
Helical
More than one strand
10 base pairs per complete turn

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-34

Erwin Chargaffs Experiment

Chargaff pioneered many of the biochemical
techniques for the isolation, purification and
measurement of nucleic acids from living cells
It was already known then that DNA contained the
four bases: A, G, C and T

Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction or display

9-35

Figure 9.14

9-37

Figure 9.14

9-38

The Data

Interpreting the Data

The compelling observation was that
Percent of adenine = percent of thymine
Percent of cytosine = percent of guanine
the percentage of C + G does not necessarily equal the
percentage of A + T

This observation became known as Chargaffs rule

It was crucial evidence that Watson and Crick used to
elucidate the structure of DNA

Watson and Crick

Familiar with all of these key observations, Watson
and Crick set out to solve the structure of DNA
They tried to build ball-and-stick models that incorporated
all known experimental observations

A critical question was how the two (or more strands)

would interact
An early hypothesis proposed that the strands interact
through phosphate-Mg++ crosslinks

!This hypothesis was, of course, incorrect

Watson and Crick

They went back to the ball-and-stick units
They then built models with the
Sugar-phosphate backbone on the outside
Bases projecting toward each other

They first considered a structure in which bases form

H bonds with identical bases in the opposite strand
ie., A to A, T to T, C to C, and G to G

Model building revealed that this also was incorrect

Watson and Crick

They then realized that the hydrogen bonding of
and T resembled that between C and G
So they built ball-and-stick models with AT and CG
interactions
These were consistent with all known data about DNA structure

Refer to Figure 9.16

Watson, Crick and Maurice Wilkins were awarded

the Nobel Prize in 1962
Rosalind Franklin died in 1958, and Nobel prizes are not
awarded posthumously

Fig. 9.16

The DNA Double Helix

Two strands are twisted together around a
common axis
There are 10 bases per complete twist
The two strands are antiparallel
One runs in the 5 to 3 direction and the other 3 to 5

The helix is right-handed

As it spirals away from you, the helix turns in a
clockwise direction

Key Features
Two strands of DNA form a
right-handed double helix.

2 nm
P

H2 N
O

O
H
N
O P O CH2
O
O
HH
HH

O
H
HH
N

O
N
O
O P O CH2 O
N
NH2
O
HH H
HH
H
H
H N
O

O
O P O CH2 O
N
O
H
H
H H
H
OH H

H
N

N
O

HH
O

H2 N

HO

H HH

NH22
N NH

3end

3end

H
H

A S
P S
hydrogen bond according to the
G C P
P S
AT/GC rule.
S
P
5phosphate
S
G
C
The 2 strands are antiparallel
S
P
with regard to their 5to 3
C
S
S
P
directionality.
PS
P
There are ~10.0 nucleotides in
S S P
each strand per complete 360
A T S P
turn of the helix.
G C S P
S
P
C G S
S P
S T A P
P S
P
3hydroxyl
P SG
One complete
P S
C G P
turn 3.4 nm
S
P
T A
S
P
S G C
S
One nucleotide
A T SP
S
P
0.34 nm
S
P
P
SC G
S P
S C G
P

The bases in opposite strands

5end

HH
O

O
O
P O
CH2
O

O
CH2 O P O
O

H
HH
N
N

HH
O
H

O
CH2 O P O
O

H2 N

CH3

5 end

Minor
groove

Major
groove

Minor
groove
Major
groove

Ball-and-stick model of DNA

Space-filling
model of DNA

The DNA Double Helix

General structural features
The double-bonded structure is stabilized by
1. Hydrogen bonding between complementary bases
A bonded to T by two hydrogen bonds
C bonded to G by three hydrogen bonds

2. Base stacking
Within the DNA, the bases are oriented so that the flattened
regions are facing each other

The DNA Double Helix

General structural features
There are two asymmetrical grooves on the
outside of the helix
1. Major groove
2. Minor groove
Certain proteins can bind within these grooves
They can thus interact with a particular sequence of bases

9-48

9-49

The Three-Dimensional Structure

of DNA
To fit within a living cell, the DNA double helix must
be extensively compacted into a 3-D conformation
This is aided by DNA-binding proteins

DNA wound
around histone
proteins

RNA Structure
The primary structure of an RNA strand is much
like that of a DNA strand
RNA strands are typically several hundred to
several thousand nucleotides in length
In RNA synthesis, only one of the two strands of
DNA is used as a template

one gene = one RNA

molecule
DNA

Nucleic acid
composed of
nucleotides
Double-stranded
Deoxyribose=sugar
Thymine
Template for
individual

RNA

Nucleic acid
composed of
nucleotides
Single-stranded
Ribose=sugar
Uracil
Many different roles!

RNA plays many roles in the cell

1.
2.
3.
4.
5.
6.
7.
8.

pre-mRNA=precursor to mRNA, newly transcribed and

not edited
mRNA= the edited version; carries the code from DNA
that specifies amino acids
tRNA= carries a specific amino acid to ribosome based
on its anticodon to mRNA codon
rRNA= makes up 60% of the ribosome; site of protein
synthesis
snRNA=small nuclear RNA; part of a spliceosome. Has
structural and catalytic roles
srpRNA=a signal recognition particle that binds to
signal peptides
RNAi= interference RNA; a regulatory molecule
ribozyme= RNA molecule that functions as an enzyme

 Although usually single-stranded, RNA molecules

can form short double-stranded regions
This secondary structure is due to complementary basepairing
A to U and C to G

This allows short regions to form a double helix

Different types of RNA secondary structures are

possible
Refer to Figure 9.23

Complementary regions
Held together by
hydrogen bonds

Noncomplementary regions
Have bases projecting away
from double stranded regions

Also called
hair-pin

 Many factors
contribute to the
tertiary structure of
RNA

Molecule contains
single- and doublestranded regions

For example
Base-pairing and
base stacking within
the RNA itself
Interactions with
ions, small
molecules and large
proteins

These spontaneously
interact to produce
this 3-D structure

Figure 9.24

Figure depicts the tertiary structure of tRNAphe

The transfer RNA that carries phenylalanine

DNA replication
Understand the basic rules
governing DNA replication
Introduce proteins that are typically
involved in generalised replication

 `It has not escaped our notice that

the specific pairing we have
postulated immediately suggests a
possible copying mechanism for
the genetic material
Watson & Crick
Nature (1953)

Original drawing by Francis Crick

Four requirements for DNA to be genetic material

carry information
Cracking the genetic code

Must replicate
DNA replication

Must allow for information to change

Mutation

Must govern the expression of the

phenotype Must
Gene function

DNA stores information in the sequence of its bases

Much of DNAs sequence-specific information is

accessible only when the double helix is unwound
Proteins read the DNA sequence of nucleotides as the
DNA helix unwinds.
Proteins can either bind to a DNA sequence, or initiate
the copying of it.
Some genetic information is accessible even in intact,
double-stranded DNA molecules
Some proteins recognize the base sequence of DNA
without unwinding it (One example is a restriction
enzyme).

DNA Replication
Process of duplication of the entire genome
prior to cell division
Biological significance
extreme accuracy of DNA replication is
necessary in order to preserve the integrity
of the genome in successive generations

In eukaryotes , replication only occurs during the S phase of

the cell cycle.

Replication rate in eukaryotes is slower

resulting in a higher fidelity/accuracy of
replication in eukaryotes

Basic rules of replication

A. Semi-conservative
B. Starts at the origin
C. Synthesis always in the 5-3
direction
D. Can be uni or bidirectional
E. Semi-discontinuous
F. RNA primers required

DNA replication
3 possible
models

Semiconservative
replication:
One strand of
duplex passed
on unchanged to
each of the
daughter cells.
This 'conserved'
strand acts as a
template for the
synthesis of a
new,
complementary
strand by the

How do we know that DNA replication is

semiconservative?
Meselson-Stahl experiments

B) Starts at origin

Initiator proteins identify specific base

sequences on DNA called sites of origin
Prokaryotes single origin site E.g E.coli oriC
Eukaryotes multiple sites of origin
(replicator)
E.g. yeast - ARS (autonomously replicating
sequences)
Prokaryotes

Eukaryotes

In what direction does DNA replication occur?

C) Synthesis is ALWAYS in the 5-3 direction
What happens if a
base mismatch
occurs?

Where does energy for addition

of nucleotide come from?

Why does DNA replication only occur in the 5 to 3 direction?

Should be PPP here

D) Uni or bidirectional

Replication forks move in one or opposite directions

E) Semi-discontinuous replication
Anti parallel strands replicated simultaneously
Leading strand synthesis continuously in 5 3
Lagging strand synthesis in fragments in 5-3

Semi-discontinuous replication

New strand synthesis always in the 5-3

direction

F) RNA primers required

Core proteins at the replication

fork
Topoisomerase s
Helicases
Primase
Single strand
binding proteins

DNA polymerase
Tethering
protein DNA ligase

Prevents torsion by DNA

breaks
separates 2 strands
RNA primer synthesis
prevent reannealing
of single strands
synthesis of new strand
stabilises polymerase
seals nick via
phosphodiester linkage

The mechanism of DNA replication

Arthur Kornberg, a Nobel prize winner

and other biochemists deduced steps
of replication
Initiation
Proteins bind to DNA and open up double helix
Prepare DNA for complementary base pairing
Elongation
Proteins connect the correct sequences of nucleotides
into a continuous new strand of DNA
Termination
Proteins release the replication complex

The mechanism of DNA

replication

Core proteins at the replication fork

Nature (2003) vol 421,pp431-435

Figure in Big Alberts t

What kind of enzyme

synthesizes the new DNA
strand?
1) RNA polymerase
2) DNA Polymerase
3) Primase
4) Helicase
5) Topoisomerase

Eukaryotic chromosomes have

multiple origins of replication
1. True
2. False

In what direction is the newly

synthesized DNA produced?
1.

5'-3'

2.

3'-5'

3.

In the direction of the major groove

4.

Both 5'-3' and 3'-5' depending on which

strand is being replicated

Nucleotides are always added to the growing

DNA strand at the 3 end, at which the DNA has a
free ______ on the 3 carbon of its terminal
deoxyribose.
1.
2.
3.

Phosphate group
Hydroxyl group
Nitrogen base

4.

Methyl group

The E. coli chromosome has 4.7x106

bp; a bi-directional replication fork
progresses at about 1000
nucleotides/sec. Therefore, the
minimum time required to complete
replication is
1) 12 min.
2) 24 min.
3) 39 min
4) 78 min
5) 120 min

What is the sequence (1 to 6) in which

these proteins function during DNA
replication

____ RNA primase

____ DNA ligase
____ DNA polymerase
____ Topoisomerase
____ DNA helicase
____ tethering proteins

Why is an RNA primer necessary for

DNA replication?
A. The RNA primer is necessary for the activity of
DNA ligase.
B. The RNA primer creates the 5 and 3 ends of the
strand.
C. DNA polymerase can only add nucleotides to
RNA molecules.
D. DNA polymerase can only add nucleotides to an
existing strand