Download Lecture 2 DNA Structure

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CHAPTER 2
Molecular Genetics DNA
Structure
In the following chapter. We will explore
the molecular structure and function of the
genetic material. We will follow this plan:
1. Nucleic Acids: Review of the Basics
2. Nucleic Acids, the research behind the
discovery of the DNA molecule structure
and function.
3. DNA structure revisited, beyond the
basics.
4. Chromosome formation and structure in
prokaryotes and eukaryotes.
Nucleic Acids: Review of the Basics
The composition and Structure of
DNA and RNA
• DNA and RNA are polymers of monomers
called nucleotides.
• A nucleotide consists of three distinct parts:
¾A pentose sugar
(deoxyribose or ibose).
¾A phosphate group PO4-3 in
the form of a triphosphate.
¾A nitrogen base
The pentose sugar
ƒ Pentose = C5
ƒ Each carbon is numbered clockwise beginning at 12:00.
ƒ Only four carbons are used in the cycle which is enclosed with an
ester bond ( -O-).
ƒ The fifth carbon is part of the cycle. The difference between
deoxyribose and ribose is that ribose has one hydroxyl group
located on Carbon #2 (C5H10O5) whereas deoxyribose does not
have a hydroxyl group having instead a H (C5H10O4)
The Phosphate Group
• There are three phosphate groups in a free
nucleotide. These three phosphates are bond
together by two high energy bonds.
• One of the terminal phosphates forms a bond
with the pentose’s carbon # 3. The phosphate
will be used for binding different nucleotides
during DNA synthesis.
The nitrogen bases
• The are two types of
nitrogen bases
• Purines: Double cycle
nitrogen bases:
Adenine and Guanine
• Pyrimidines: Single
cycle nitrogen bases:
Cytosine, Thymine
and Uracil.
• The dNTPs (deoxyribonucleotides tri
phosphates) nucleotides are bond together by an
endergonic dehydration synthesis reaction.
Water
• dNTP + dNTP
• The reaction leads to
the formation of a
Phosphodiester bond
between C3 of one
nucleotide and C5 of the
other nucleotide.
dNTP-dNTP
• The addition of multiple
nucleotides using
deshydration synthesis
lead to the formation of
a large chain.
• Notice that the first
nucleotide (C5) has a
phosphate and the last
nucleotide (C3) does
not. Thus this strand of
DNA “runs” from a 5’
3’ and it is known
as the “sense” strand.
The second strand
• The second strand of DNA follows the same
principles that we have studied so far. It is
positioned “upside down” and it is a mirror image
of the first strand (a 180 degree vertical rotation).
• However, if this was a “true” mirror image, the
nitrogen bases should be the same and that is
not the case: The nitrogen bases are
complementary.
• Complementary bases mean, that if in the first
strand we find a purine base, in the second
strand we will find a pyrimidine and viceversa.
Complementary bases
First strand (sense)
Second strand (antisense)
ƒThe two strands are
linked together by
hydrogen bonds.
ƒThere are two
hydrogen bonds
between thymine and
adenine and three
hydrogen bonds
between cytosine and
guanine.
ƒThe C-G pair bond
is slightly stronger
than the A-T
Once the two strands are bond together, the entire
structure twists to the right (right handed or clock
wise) to form a helical structure known as a “double
helix”.
Nucleic Acids, the research behind the discovery
of the DNA’s structure and function.
• 1868 Frederich Miescher Isolated “nuclein”
from the nuclei of cells. Nuclein is now
known as DNA.
• 1911. T.H. Morgan’s group showed that
genes are located on chromosomes.
• 1928 Frederick Griffith discovered genetic
transformation of a bacterium and called
the agent responsible the “transforming
principle.”
• 1941 Avery. MacLeod and McCarty
showed that Griffith transforming principle
• 1948 Edwin Chargaff produced Chargaff rule: The amount of
A=amount of T and the amount of C=amount of G.
• 1952 Alfred Hershey and Martha Chase demonstrated that DNA
was the genetic material.
• 1950’s Rosaling Franklin and Maurice Wilkins provided
photographs of X-ray diffractions and provided physical
information about DNA: A double helix and their measurents.
• 1953 In a leap of imagination James Watson and Francis Crick
produced the double helix model of the molecule of DNA.
• 1956Gierer and Schramm, and 1957 Fraenkel-Conrat and Singer
concluded that RNA was the genetic material of some viruses.
The search for genetic material
• Once T.H. Morgan’s group showed that genes are
located on chromosomes (1911), the two
constituents of chromosomes - proteins and DNA
- were the candidates for the genetic material.
• The discovery of the genetic role of DNA began
with research by Frederick Griffith in 1928.
• He studied Streptococcus pneumoniae, a bacterium that
causes pneumonia in mammals. He used two
strains:
The R strain was harmless.
The S strain was pathogenic.
• The experiment was as follows. He injected:
S bacteria
Mouse dies
Type III S bacteria
were isolated
from the mouse
R bacteria
Heat killed
S bacteria
Live R +Heat killed
S bacteria
Mouse survives Mouse survives Mouse dies
No living bacteria
were isolated
from the mouse
No living bacteria
were isolated
from the mouse
Type III S bacteria
were isolated
from the mouse
Live R +Heat killed
S bacteria
ƒThe mouse died and he recovered the
pathogenic strain from the mouse’s
blood.
ƒThe heat had killed all the molecules but
not “the information to make capsules”.
The R bacteria had assimilated this
material from the dead S bacteria.
ƒGriffith called this phenomenon
Type III S bacteria
were isolated
from the mouse
transformation, a change in genotype
and phenotype due to the assimilation of
a foreign substance (now known to be
DNA) by a cell. The substance, he
called “transforming principle”.
Avery. MacLeod and McCarty showed that
Griffith transforming principle was DNA.
Allow sufficient time for the DNA to be taken up by the type R bacteria. Add an antibody that
aggregates R bacteria that has not been transformed and remove
by centrifugation. Plate the remaining (the transformed bacteria) on petri dishes.
The Hershey and Chase
Experiment
• In 1952, Alfred Hershey and Martha Chase showed that
DNA was the genetic material of the phage T2.
• The T2 phage, consisting almost entirely of DNA and
protein, attacks Escherichia coli (E. coli), a common
intestinal bacteria of mammals.
• This phage can quickly
turn an E. coli cell into
a T2-producing factory
that releases phages
when the cell ruptures.
Bacteriophage T2 lytic life cycle
• To determine the source of genetic material in the
phage, Hershey and Chase designed an experiment
where they could label protein or DNA and then
track which entered the E. coli cell during infection.
– They grew one batch of T2 phage in the presence of
radioactive sulfur, marking the proteins but not DNA.
– They grew another batch in the presence of radioactive
phosphorus, marking the DNA but not proteins.
– They allowed each batch to infect separate E. coli
cultures.
– Shortly after the onset of infection, they spun the
cultured infected cells in a blender, shaking loose any
parts of the phage that remained outside the bacteria.
– The mixtures were
spun in a centrifuge
which separated the
heavier bacterial cells
in the pellet from
lighter free phages
and parts of phage in
the liquid supernatant.
– They then tested the
pellet and supernatant
of the separate
treatments for the
presence of
radioactivity.
• Hershey and Chase found that when the bacteria had
been infected with T2 phages that contained radio-labeled
proteins, most of the radioactivity was in the supernatant,
not in the pellet.
• When they examined the bacterial cultures with T2 phage
that had radio-labeled DNA, most of the radioactivity was
in the pellet with the bacteria.
• Hershey and Chase concluded that the injected DNA of
the phage provides the genetic information that makes the
infected cells produce new viral DNA and proteins, which
assemble into new viruses.
The Chargaff Rule
• 1948 Edwin Chargaff hydrolized DNA from
different organisms and demonstrated that the
composition of double stranded DNA was 50%
purine and 50% pyrimidine.
A+T =1
C+G
• Furthermore, he demonstrated that the
amount of Adenine was very similar to the
amount of Thymine and the amount of
Guanine was similar to the amount of
Cytosine. These equivalencies are known as
Chargaff rules.
Watson and Crick discovered the
double helix by building models to
conform to X-ray data
• By the beginnings of the 1950’s, the race
was on to move from the structure of a single
DNA strand to the three-dimensional
structure of DNA.
• Among the scientists working on the problem
were Linus Pauling, in California, and
Maurice Wilkins and Rosalind Franklin, in
London.
• Maurice Wilkins and Rosalind Franklin used
X-ray crystallography to study the structure
of DNA.
– In this technique, X-rays are diffracted as they
passed through aligned fibers of purified DNA.
– The diffraction pattern can be used to deduce
the three-dimensional shape of molecules.
Franklin concluded that DNA was helical
structure with two distinctive regularities
(grooves) of 0.34 nm and a 3.4 nm along its
axis.
The Watson and Crick Model
Watson and
Crick used the
data produced
by Wilkins and
Franklin to
produce a three
dimensional
model of the
structure of
DNA.
The key breakthrough came when
Watson put the sugar-phosphate
chain on the outside and the
nitrogen bases on the inside of the
double helix.
The sugar-phosphate chains of each
strand are like the side ropes of a
rope ladder, pairs of nitrogen bases,
one from each strand, form rungs
and the ladder forms a twist every
ten bases.
And the rest is History!
One more time, let’s revise the chemical structure
and physical characteristics of the molecule of
DNA.
Double Stranded DNA Can Occur in Three Conformations
A-DNA is the dehydrated form..
It is not usually found in cells. It is
a right-handed helix with
10.9bp/turn, with the bases
inclined 13° from the helix axis. ADNA has a deep and narrow
major groove, and a wide and
shallow minor groove.
A-DNA
B-DNA
Z-DNA
Double Stranded DNA Can Occur in Three Conformations
A-DNA
B-DNA
B-DNA is the hydrated form of
DNA, the kind normally found in
cells. It is also a right-handed helix,
with only 10.0bp/turn, and the
bases inclined only 2° from the
helix axis. B-DNA has a wide
major groove and a narrow minor
groove, and its major and minor
Z-DNA grooves are of about the same
depth.
Double Stranded DNA Can Occur in Three Conformations
A-DNA
B-DNA
Z-DNA is a left-handed helix with
a zigzag sugar-phosphate
backbone that gives it its name. It
has 12.0bp/turn, with the bases
inclined 8.8° from the helix axis.
Z-DNA has a deep minor groove,
and a very shallow major groove.
Its existence in living cells has not
Z-DNA been proven.
DNA is organized in Chromosomes
• 1. Cellular DNA is organized into
chromosomes. A genome is the chromosome or
set of chromosomes that contains all the DNA
of an organism.
• 2. In prokaryotes the genome is usually a single
circular chromosome. In eukaryotes, the genome
is one complete haploid set of nuclear
chromosomes; mitochondrial and chloroplast
DNA are not included.
Viral Chromosomes
• A virus is nucleic acid surrounded by a protein
coat. The nucleic acid may be dsDNA, ssDNA,
dsRNA, or ssRNA, and it may be linear or
circular, a single molecule or several segments.
For example:
• The T-even phages (T2, T4, and T6) have
dsDNA in one linear DNA molecule.
• φFX174 is a small, simple virus with one short
ssDNA chromosome
• Bacteriophage l chromosome’s is a linear
molecule of dsDNA, but after the virus infects its
host, the chromosome becomes circular.
Prokaryotic Chromosomes
• The typical prokaryotic
genome is one circular
dsDNA chromosome, but
some prokaryotes have a
main chromosome and one
or more smaller ones. The
smaller ones are called
plasmids.
Both Eubacteria and Archaebacteria lack a membranebounded nucleus, and their DNA is densely arranged in a
cytoplasmic region called the nucleoid.
„
• If E. coli was gently
lysed, it would releases
one 4.6-Mb circular
chromosome, highly
supercoiled. A 4.6-Mb
double helix is about
1mm in length, about 103
times longer than an E.
coli cell. DNA
supercoiling helps it fit
into the cell.
• Supercoiling is “super
twisting” the DNA. It is
DNA
length is
about
1000 x
cell
length
ƒProkaryotes also
organize their DNA
into looped domains,
with the ends of the
domains held so that
each is supercoiled
independently.
ƒThe compaction
factor for looped
domains is about 10fold. In E. coli there
are about 100 domains
of about 40kb each.
Eukaryotic chromosomes
• Most eukaryotes have a diploid number of
chromosomes.
• Eukaryotic chromosomes are made of chromatin,
a combination of DNA and proteins, and its levels
of condensation varies during the different stages
of the cell life cycle. When looked with an
electron microscope, chromatin shows a structure
of “beads-on-a-string” structure. These “beads”
are called” nucleosomes.
• These nucleosomes are
the units of chromatin
formed by the proteins and the DNA.
• Histones are abundant, small proteins with a net
(+) charge. The five main types are H1, H2A,
H2B, H3, and H4.
• Two molecules of each histone H2, H2A, H3
and H4 group together to form the “histone
core” and the DNA wraps around 1.65 times
(about 147 bp fragment). This is called a
nucleosome.
Nucleosome cores
are about 11 nm in
diameter.
ƒThe function of the histones is to condense the DNA.
The nucleosome core provides a 7-fold condensation
factor. The DNA between nucleosomes is called Linker
DNA. The size of the DNA linker varies within and among
organisms.
ƒH1 further
condenses the
DNA by
connecting
nucleosomes to
create chromatin
with a diameter of
30nm, for an
additional 6-fold
condensation.
ƒThis is called the 30 nm chromatin
fiber.
ƒ The formation of this 30 nm chromatin
fiber is explained by the solenoid model.
This model proposes that the
nucleosomes form a spiral with 6
nucleosomes per turn.
ƒFurther packing of the chromatin fibers is less understood.
It is believed that loops of DNA attached themselves to
non-histone proteinaceous “scafolds”. In cross section it
seems like the loops are arranged like the petals of a flower
around the “scafold”.
ƒAn average human chromosome has about 2000 domains
(loops).
Summary of the
condensation
and coiling of
chromatin in
Eukaryotic
chromosomes.
A methaphasic
chromosome is
400 times
thicker than the
naked DNA.
• Eukaryotic chromosomes levels of condensation
varies during the different stages of the cell life
cycle. The most disperse is when the DNA is
going to duplicate (S phase) and the most
condensed is during metaphase of Mitosis and
Meiosis.
• There are two types of chromatin: Euchromatin
and Heterochromatin.
• Euchromatin condenses and decondenses with
the cell cycle. It is actively transcribed, and lacks
repetitive sequences. Euchromatin accounts for
most of the genome in active cells.
• Heterochromatin remains condensed throughout the
cell cycle. It replicates later than euchromatin, and is
transcriptionally inactive. There are two types based on
activity:
• Constituitive heterochromatin: occurs at the same sites
in both homologous chromosomes of a pair, and
consists mostly of repetitive DNA (e.g., centromeres).
• Facultative heterochromatin varies between cell types
or developmental stages, or even between homologous
chromosomes. It contains condensed, and thus
inactive, euchromatin (e.g., inactivated Xchromosomes
or Barr bodies).
• Centromeres and
telomeres are
eukaryotic
chromosomal regions
with special functions.
• Centromeres are the
site of the
kinetochore, where
spindle fibers attach
during mitosis and
meiosis. They are
required for accurate
segregation of
chromatids.
• The telomeres are usually the “ends” of
the chromosome (the fragment of DNA at
the ends).
• Telomeres are needed for chromosomal
replication and stability. Generally
composed of heterochromatin, they
interact with both the nuclear envelope
and each other. All telomeres in a species
have the same sequence.
• The total amount of DNA in the haploid
genome of a species is its C value .
• A genome is the information in one
complete haploid chromosome set.