Biology 3000
Molecular Genetics
Nature of the genetic material
Isolation of novel substance, nuclein: Friedrich Miescher, 1869,
Tübingen, Germany
DNA and RNA had been separated from the protein that clings to
them in the cell: end of 19th century
DNA and RNA nucleotide composition: Levene and Jacobs, 1930th
• DNA and RNA are composed of four nitrogen-containing bases
• DNA and RNA differ in sugar content (deoxyribose vs. ribose)
• Each nucleotide is composed of nitrogenous base coupled with a
sugar-phosphate
The experiment of F. Griffith

The killed virulent bacteria could transform the avirulent strain to virulent
Conclusions:
Virulent trait was passed from the dead cells to alive.
Transformation was stable. The newly acquired by avirulent
strain virulence (the ability to kill animal cells) was passed to
descendents as a heritable trait.
Avirulent cells gained the gene for virulence.
Transforming substance – the gene of virulence itself.
The chemical nature of transforming substances???
DNA, RNA or protein?
1944 – Oswald Avery, Colin MacLeod and Maclyn McCarty defined the
chemical nature of transforming substance
1. Removing proteins from the extract with organic solvents
has no effect on transformation
2. Treatment with trypsin and chemotrypsin: had no effect
on transformation
3. Ribonuclease treatment: had no effect on transformation
Neither protein nor RNA were the transforming substance
 Treatment with DNase destroyed the transformation ability
of the extract (Avery et al.)
Physical-chemical analysis to support the hypothesis
Avery et al.
1. Ultracentrifugation – TS has high molecular mass.
2. Electrophoresis – TS has high mobility, because of
high charge-to–mass ratio.
3. UV absorption spectrophotometry – absorption
spectrum of TS matched that of DNA – 260nm.
4. Elementary chemical analysis – TS has low (1.67)
N/P ratio. Similar for DNA that is rich in both P and N.
DNA or Protein?
DNA is a monotonous repeat of four nucleotides??
•
•
nitrogenous bases are not found in equal proportions in DNA
various species differ in a base composition of DNA (Chargaff E., 1950)
Protein contamination in TS??
•
purification of TS to 0.02% of protein (Hotchkiss R.)
 DNA can transform cells
 DNA can carry other genetic traits
1952 – Hershey and Chase Experiment
Phage infection – phage enters the cell and directs the
synthesis of new phage particles
Phage has DNA and protein only
Do the genes reside in protein or DNA?
Different labeling –
•
•
32P
35S
for DNA (DNA is reach in phosphorus and has no sulfur)
for protein (Phage protein contains sulfur and has no phosphorus)
What went in?

Infected cells contained 32P labeled DNA
DNA 3D structure
Chargaff’s Experiments: in various species the content of
purines is equal to the content of pyrimidines
• Chargaff’s rule: in double stranded DNA molecule globally
%A=%T and %G=%C
Wilkins and Franklin: X-ray diffraction analysis
• At specific relative humidity of the air the DNA fiber is enough like
a crystal – it can diffract X-rays in interpretable way
Watson and Crick: a double helix model of DNA
• DNA double helix is uniform
• Sugar-phosphate backbones are on the outside
and bases on the inside
• Bases are paired; purine in one strand always pairs
with pyrimidine in the other
The native state of the DNA is a double helix of two
anti-parallel chains with complementary nucleotide
sequences
DNA consists of two associated antiparallel polynucleotide
strands that wind together through space in helical fashion
which is often described as double helix.
Chemical structure of principal bases in nucleic acids
The bases adenine (A), guanine (G), and cytosine (C ) are found in both
DNA and RNA, thymine (T) is found only in DNA and uracil (U)– only in
RNA.
The nucleotides are either purines (A, G), or pyrimidines (T, C, U)
Nitrogenous bases
Replacement of thymine for uracil in DNA (Thymine = methyl-Uracil)
•
protects DNA form attack by foreign nucleases (enzymes that break down DNA
and RNA)
•
maintains fidelity of DNA replication (methyl-group restricts uracil (thymine) to
pairing only with adenine)
•
prevents accumulation of C to U mutations in DNA
Sugars of nucleic acids
Nucleosides
Cells and extracellular fluids contain small concentrations of
building blocks – nucleosides – combinations of a base and sugar
without phosphate.
Nucleotides which have one, two or three attached phosphate
groups are referred as nucleoside phosphates.
The nucleoside phosphates that are used as building blocks are
esterified at the 5’hydroxyl.
Supply of nucleoside triphosphates is necessary for the
synthesis of nucleic acids.
These compounds also have other functions in the cells. ATP
is the most widely used energy carrier.
DNA
 Nucleotides are joined together with phosphodiester bonds that involve
phosphoric acid linked to two sugars - one through sugar 5’ group, another
through the 3’ group.
 Strands are held together by cooperative energy of many hydrogen
bonds in addition to hydrophobic interactions
Two strands of DNA are antiparallel
Outside of DNA the spaces between
intertwined strands from 2 helical
grooves – major groove and minor
groove
DNA – forms of double helix
3’
5’ 5’
3’
5’
3’ 3’
5’
Right-handed
double helix
Left-handed
double helix
The x-ray diffraction patterns
indicated that the stacked bases
are regularly spaced 0.34 nm
apart along the helix axes.
Indeed, helix makes turn in every
3.4 nm (34A), thus - 10 bases per
turn –this is called B form of DNA
A and B forms of DNA
A-form – has 11 bases per turn and one turn is about
31A. - Pitch is about 28A. It is more compact. Plane
bp is not any more perpendicular – tilts 20o.
On the outside
the B molecule,
the spaces
between intertwined strands
form two helical
grooves of
different widths –
major and minor
groove.
Consequently,
each base is
accessible from
outside the helix
to the molecules
that bind to DNA
by contacting
chemical groups
within the
grooves.
Z and B forms of DNA
Some short molecules can adopt an
alternative left-handed confirmation.
It is called Z DNA because the bases
seem to zigzag when viewed from side.
It is unknown if this form occurs in
nature. If yes, they could be a
recognition signal in regions of DNA.
Seldom, triple helical confirmation:
Z DNA
B DNA
DNA can bend
The hydrogen and hydrophobic bonds between polynucleotide strands provide
stability to DNA. However, the double helix is flexible about its long axis,
because there are no hydrogen bonds between successive residues of a
strand. This property allows DNA to bend
It is also believed that particular regions of DNA, especially that bound to the
protein depart from standard structure.
The sequence-dependant deformation of DNA about the long axis – creating
bent DNA is now recognized in naked DNA without proteins, as well as in cases
in which DNA in combination with proteins is bent.
For example: the adenilate residues, if all are in one strand, in A-T rich DNA
tend to stack at greater angle to the helix axis than thymidilate (T) residues. This
leads to bending DNA even without proteins bound.
Binding of repressor protein encoded by bacteriophage 434 results in bend of
DNA. Binding of repressor protein to viral genome prevents its transcription by
host cell enzymes
TATA box binding protein (TBP) –
causes complete change in winding
and direction of the double helix.
Transcription requires the binding
of TBP
Two strands can separate, causing DNA to denature
Unwinding and separation of the strands can be induced experimentally:
 By heating –→the strands separate→ DNA is said to be denatured.
Melting temperature – Tm – the temperature at which 50% of DNA molecule melts
Hyperchromic shift: the
increase in a DNA
solution’s absorbance of
260-nm light on
denaturation
• A-T rich regions melt faster than G-C rich regions
• solutions of low ion concentrations, high pH destabilize double helix
• DNA is also denatured by exposure to agents that destabilize hydrogen
bonds, such as alkaline solutions and concentrated solutions of formamide
and urea.
DNA renaturation
Single stranded molecules that result from denaturation form random
coils without regular structure.
3’
5’
5’
3’
t, OH-
5’
3’
Native state
3’
5’
Renaturation
(special conditions
required)
3’
5’
Single-stranded
denatured state
5’
3’
Renatured state
By lowing the temperature and increasing the ion concentration, the two
complementary strands in time will renature and form a perfect double helix.
Fast cooling prevents renaturation and DNA remains denaturated
Denaturation and renaturation of DNA are the basis of nucleic acid
hybridization
Factors that contribute to renaturing efficiency:
1. Temperature – the best T of renaturing is 25o lower than Tm. Low
enough not to promote denaturing, high enough to allow rapid
diffusion of DNA molecules and to weaken transient bonding
between mismatched sequences and short intrastrand base-paired
regions.
Rapid cooling prevents renaturing.
2. DNA concentration – within reasonable limits – the higher the
concentration the faster annealing.
3. Renaturing time – the longer the better.
4. DNA complexity.
Many DNA molecules are circular
Where:




Bacteria and viruses (all bacterial and many viral DNAs are circular)
Almost in mitochondria of eukaryotic cells
In chloroplasts of plant cells
In some single-cell eukaryotes
DNA can be supercoiled
Denaturation of circular DNA
NB: nicking of circular DNA can occur naturally during replication or experimentally by
cleaving a single phosphodiester bond in the circle with deoxyribonuclease (DNA degrading
enzyme).
If both strands are closed circles,
heat
denaturation disrupts double helix, but
two single strands become tangled
cool
about each other and can not separate.
Double stranded native molecule
Tangled strands
If one or both strands are nicked,
the two strands will separate
heat
completely
cool
Nick in one strand
Strands completely
separated
Gene Functions
1.Storing Information
•
DNA is a “cellular library” that contains all the information
required to build the cells and the tissues of an organism.
Genetic information is arranged in genes, hereditary units
controlling specific traits of an organisms.
2. Replication
•
Precise duplication of DNA allows passage of genetic
information unchanged from generation to generation.
3. Mutation
•
Accumulation of spontaneous and induced changes in
nucleotide sequence of DNA provide a raw material for evolution
The Central Dogma of Molecular Biology

NB: Some RNA viruses
(retroviruses, ex. HIV) synthesize
double stranded DNA from single
stranded RNA template in a
process called reverse
transcription

NB: Some RNA viruses can
copy information directly from
one RNA to another in a process
of RNA replication
Francis Crick, 1958; Nature 1970
Reading:
R. Weaver, Molecular Biology, 4th ed.
Chapter 2: pages 13-30.
Chapter 3: pages 31-38; 46-50.