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1/28/2011
Biology Review
Cell Structure
Biological Molecules (DNA & Proteins)
Central Dogma
Structure and Function of DNA
Replication, Transcription, Translation
Regulation of Gene Expression
Prokaryotic cell
Eukaryotic cell
Membrane
DNA
(no nucleus)
Membrane
Cytoplasm
Organelles
Nucleus (contains DNA)
1 µm
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Prokaryotic cell
Fimbriae
Cell wall
Circular chromosome
Capsule
Sex pilus
Internal
organization
Flagella
Nuclear
envelope
ENDOPLASMIC RETICULUM (ER)
Flagellum
Rough ER
NUCLEUS
Nucleolus
Smooth ER
Chromatin
Centrosome
Plasma
membrane
CYTOSKELETON:
Microfilaments
Intermediate
filaments
Microtubules
Ribosomes
Microvilli
Golgi
apparatus
Peroxisome
Mitochondrion
Lysosome
Animal cell
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Structure
Cell Component
Nucleus
The eukaryotic cell’s genetic
instructions are housed in
the nucleus and carried out
by the ribosomes
Function
Surrounded by nuclear
envelope (double membrane)
perforated by nuclear pores.
The nuclear envelope is
continuous with the
endoplasmic reticulum (ER).
Houses chromosomes, made of
chromatin (DNA, the genetic
material, and proteins); contains
nucleoli, where ribosomal
subunits are made. Pores
regulate entry and exit os
materials.
Two subunits made of ribosomal RNA and proteins; can be
free in cytosol or bound to ER
Protein synthesis
(ER)
Ribosome
Cell Component
The endomembrane system
regulates protein traffic and
performs metabolic functions
in the cell
Structure
Extensive network of
Endoplasmic reticulum
membrane-bound tubules and
(Nuclear sacs; membrane separates
envelope)
lumen from cytosol;
continuous with
the nuclear envelope.
Golgi apparatus
Lysosome
Vacuole
Stacks of flattened
membranous
sacs; has polarity
(cis and trans
faces)
Function
Smooth ER: synthesis of
lipids, metabolism of carbohydrates, Ca2+ storage, detoxification of drugs and poisons
Rough ER: Aids in sythesis of
secretory and other proteins
from bound ribosomes; adds
carbohydrates to glycoproteins;
produces new membrane
Modification of proteins, carbohydrates on proteins, and phospholipids; synthesis of many
polysaccharides; sorting of
Golgi products, which are then
released in vesicles.
Breakdown of ingested subMembranous sac of hydrolytic
stances cell macromolecules,
enzymes (in animal cells)
and damaged organelles for
recycling
Large membrane-bounded
vesicle in plants
Digestion, storage, waste
disposal, water balance, cell
growth, and protection
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Cell Component
Mitochondrion
Mitochondria and chloroplasts change energy from
one form to another
Structure
Bounded by double
membrane;
inner membrane has
infoldings (cristae)
Function
Cellular respiration
Chloroplast
Photosynthesis
Typically two membranes
around fluid stroma, which
contains membranous thylakoids
stacked into grana (in plants)
Peroxisome
Specialized metabolic
compartment bounded by a
single membrane
Contains enzymes that transfer
hydrogen to water, producing
hydrogen peroxide (H2O2) as a
by-product, which is converted
to water by other enzymes
in the peroxisome
Overview: The Molecules of Life
All living things are made up of four classes of large
biological molecules: carbohydrates, lipids, proteins, and
nucleic acids
Within cells, small organic molecules are joined together to
form larger molecules
Macromolecules are large molecules composed of
thousands of covalently connected atoms
Molecular structure and function are inseparable
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Macromolecules are polymers,
built from monomers
A polymer is a long molecule consisting of many
similar building blocks
These small building-block molecules are called
monomers
Three of the four classes of life’s organic molecules
are polymers:
Carbohydrates
Proteins
Nucleic acids
The Diversity of Polymers
Each cell has thousands of different kinds
of macromolecules
Macromolecules vary among cells of an
organism, vary more within a species, and
vary even more between species
An immense variety of polymers can be
built from a small set of monomers
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Proteins have many structures, resulting
in a wide range of functions
Proteins account for more than 50% of the
dry mass of most cells
Protein functions include structural support,
storage, transport, cellular
communications, movement, and defense
against foreign substances
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Proteins
Proteins perform many functions:
Structural roles: i.e. keratin and collagen
Muscle contraction: i.e. actin and myosin Endocrine
function: i.e. hormones
Transport molecules in the blood (hemoglobin carries
O2)
Channel proteins.
Immune function: i.e. antibodies
Biological catalysts: i.e. Enzymes
Enzymes are a type of protein that acts as a
catalyst to speed up chemical reactions
Enzymes can perform their functions repeatedly,
functioning as workhorses that carry out the
processes of life
Etc.
Proteins are made up of amino acids
Polypeptides
Polypeptides are polymers built from
the same set of 20 amino acids
A protein consists of one or more
polypeptides
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Amino Acid Monomers
Amino acids are organic molecules with
carboxyl and amino groups
Amino acids differ in their properties due to
differing side chains, called R groups
α carbon
Amino
group
Carboxyl
group
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Nonpolar
Glycine
(Gly or G)
Valine
(Val or V)
Alanine
(Ala or A)
Methionine
(Met or M)
Leucine
(Leu or L)
Trypotphan
(Trp or W)
Phenylalanine
(Phe or F)
Isoleucine
(Ile or Ι )
Proline
(Pro or P)
Polar
Serine
(Ser or S)
Threonine
(Thr or T)
Cysteine
(Cys or C)
Tyrosine
(Tyr or Y)
Asparagine Glutamine
(Asn or N) (Gln or Q)
Electrically
charged
Acidic
Basic
Aspartic acid Glutamic acid
(Glu or E)
(Asp or D)
Lysine
(Lys or K)
Arginine
(Arg or R)
The twenty amino acids of proteins.
The amino acids are grouped here
according to the properties of the
side chains (R groups) highlighted in
white. The amino acids are shown in
their prevailing ionic forms at pH 7.2
the pH within a cell. The three letter
and more commonly used one letter
abbreviations for the amino acids
are in the parentheses. All of the
amino acids used in proteins are the
same enantiomer called the L form
as shown here
Histidine
(His or H)
Amino Acid Polymers
Amino acids are linked by peptide bonds
A polypeptide is a polymer of amino acids
Polypeptides range in length from a few to
more than a thousand monomers
Each polypeptide has a unique linear
sequence of amino acids
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Fig. 5-18
Peptide
bond
(a)
Side chains
Peptide
bond
Backbone
(b)
Amino end
(N-terminus)
Making a polypeptide chain.
A) Peptide bonds formed by
dehydration reactions link
the carboxyl group of one
amino acid to the amino
group of the next. B) The
peptide bonds are formed
one at a time with the amino
acid at the amino end (N
terminus). The polypeptide
has a repetitive backbone
(purple) to which the amino
acid side chains are
attached.
Carboxyl end
(C-terminus)
Determining the Amino Acid
Sequence of a Polypeptide
The amino acid sequences of
polypeptides were first determined by
chemical methods
Most of the steps involved in sequencing
a polypeptide are now automated
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Protein Structure and Function
A functional protein consists of one or
more polypeptides twisted, folded, and
coiled into a unique shape
The sequence of amino acids
determines a protein’s threedimensional structure
A protein’s structure determines its
function
Groove
Groove
a) A ribbon model shows how the single
polypetide chain folds and coils to form
the functional protein. (the yellow lines
represent one type of chemical bond that
stabilizes the protein’s shape)
b) A space-filling model of lysozyme
Shows more clearly the globular
shape seen in many proteins as well
as the specific three dimensional
structure unique to lysosyme.
Structure of a protein, the enzyme lysozyme. Present in our sweat. Tears and
saliva lysozyme is an enzyme that helps prevent infection by binding to and
destroying specific molecules on the surface of many kinds of bacteria. The
groove is the part of the protein that recognizes and binds to the target
molecules on bacterial walls.
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Levels of protein organization
The structure of proteins has at least three levels of organization, and some can
have four
Primary structure- linear unique sequence of amino acids joined by peptide
bonds. The primary structure of a protein is its unique sequence of amino acids
Peptide bonds are polar and therefore the C=O of one amino acid can also H
bond to the N-H of another amino acid, and a water molecule is formed
Secondary structure- When the protein takes an orientation in space, a coiling
of the chain gives rise to a helix whereas a folding of the chain leads to pleated
sheets. H bonds between the peptide bonds hold the shape.
Tertiary Structure- Tertiary structure is determined by interactions among
various side chains (R groups) and consists of the final 3 D shape of the
protein. This type of structure are maintained by :
Covalent, ionic bonds between amino acid R groups
Quaternary structure- If the protein is made up of more than one polypeptide
chain i.e. hemoglobin
It is critical that proteins have a certain structure (structure function relationship)
It is crucial not to get denatured by changes in pH and temperature etc.
Primary
Structure
Secondary
Structure
Tertiary
Structure
Quaternary
Structure
β pleated sheet
+H N
3
Amino end
Examples of
amino acid
subunits
α helix
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Primary Structure
Sequence of amino acids, it is like the
order of letters in a long word
Unique for each protein, encoded by
DNA (determined by inherited genetic
information)
Two linked amino acids = dipeptide
Three or more = polypeptide
Backbone of polypeptide has N atoms:
-N-C-C-N-C-C-N-C-C-N-
1
5
+H N
3
Amino end
Primary Structure
10
Amino acid
subunits
15
1
+H
20
5
3N
25
Amino end
10
Amino acid
subunits
15
75
80
90
85
20
95
105
100
110
115
25
120
125
Carboxyl end
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Secondary Structure
Secondary structure- When the
protein takes an orientation in
space, a coiling of the chain
gives rise to a helix whereas a
folding of the chain leads to
pleated sheets.
H bonds between the peptide
bonds hold the shape
The coils and folds of secondary
structure result from hydrogen
bonds between repeating
constituents of the polypeptide
backbone
Typical secondary structures are
a coil called an α helix and a
folded structure called a β
pleated sheet
Secondary Structure
β pleated sheet
Examples of
amino acid
subunits
α helix
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Tertiary structure
Tertiary structure is the final 3 D shape of the protein.
Tertiary structure is determined by interactions between R
groups, rather than interactions between backbone constituents
These interactions between R groups include hydrogen bonds,
ionic bonds, hydrophobic interactions, and van der Waals
interactions
Strong covalent bonds called disulfide bridges may reinforce
the protein’s conformation
Tertiary Structure
Quaternary Structure
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Hydrophobic
interactions and
van der Waals
interactions
Polypeptide
backbone
Hydrogen
bond
Disulfide bridge
Ionic bond
Polypeptide
chain
β − Chains
Iron
Heme
α − Chains
Hemoglobin
Collagen
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Quaternary Structure
Quaternary structure- If the protein is
made up of more than one polypeptide
chain i.e. hemoglobin
Hemoglobin is a globular protein
consisting of four polypeptides: two alpha
and two beta chains
Collagen is a fibrous protein consisting of
three polypeptides coiled like a rope
What Determines Protein Structure?
In addition to primary structure, physical
and chemical conditions can affect
structure
Alterations in pH, salt concentration,
temperature, or other environmental factors
can cause a protein to unravel
This loss of a protein’s native structure is
called denaturation
A denatured protein is biologically inactive
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Denaturation
Normal protein
Renaturation
Denatured protein
Denaturation and renaturation of a protein. High temperatures or various
chemical treatments will denature a protein, causing it to lose its shape and
hence its ability to function. If the denatured proteins remains dissolved it can
often renature when the chemical and physical aspects of its environment are
restored to normal
Protein Folding in the Cell
It is hard to predict a protein’s structure
from its primary structure
Most proteins probably go through
several states on their way to a stable
structure
Chaperonins are protein molecules that
assist the proper folding of other
proteins
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Scientists use X-ray crystallography to
determine a protein’s structure
Another method is nuclear magnetic
resonance (NMR) spectroscopy, which
does not require protein crystallization
Bioinformatics uses computer programs to
predict protein structure from amino acid
sequences
EXPERIMENT
Diffracted
X-rays
X-ray
source X-ray
beam
Crystal
Digital detector
X-ray diffraction
pattern
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Nucleic acids store and transmit
hereditary information
The amino acid sequence of a
polypeptide is programmed by a unit of
inheritance called a gene
Genes are made of DNA, a nucleic
acid
Nucleic Acids
There are two types of nucleic acids:
DNA (deoxyribonucleic acid) stores genetic information in the cell and
organism-it replicates and gets transmitted to other cells when they
divide and also when an organism reproduces. DNA provides
directions for its own replication. DNA also directs synthesis of
messenger RNA (mRNA) and, through mRNA, controls protein
synthesis (occurs in ribosomes) therefore DNA codes for the amino
acids in proteins
RNA (ribonucleic acid) can function as an intermediary molecule which
conveys DNAs instructions regarding the aa sequence in proteins
(among many other roles)
Structure:
Both are made up of nucleotides ( a molecular complex of phosphate a
pentose sugar and a Nitrogenous base)
There are two types of nucleic acids:
Deoxyribonucleic acid (DNA)
Ribonucleic acid (RNA)
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DNA
1 Synthesis of
mRNA in the
nucleus
mRNA
NUCLEUS
CYTOPLASM
mRNA
2 Movement of
mRNA into cytoplasm
via nuclear pore
Ribosome
DNA RNA protein. In a
eukaryotic cell, DNA in the
nucleus programs protein
production in the
cytoplasm by dictating
synthesis of the
messenger RNA mRNA.
The cell nucleus is
actually much larger
relative to the other
elements in this figure
3 Synthesis
of protein
Polypeptide
Amino
acids
The Structure of Nucleic
Acids
Nucleic acids are polymers called polynucleotides
Each polynucleotide is made of monomers called
nucleotides
Each nucleotide consists of a nitrogenous base, a pentose
sugar, and a phosphate group
The portion of a nucleotide without the phosphate group is
called a nucleoside (nitrogenous base + sugar)
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5 end
Nitrogenous bases
Pyrimidines
5C
3C
Nucleoside
Nitrogenous
base
Cytosine (C)
Thymine (T, in DNA) Uracil (U, in RNA)
Purines
Phosphate
group
5C
Sugar
(pentose)
Adenine (A)
3C
Guanine (G)
(b) Nucleotide
Sugars
3 end
(a) Polynucleotide, or nucleic acid
Components of nucleic acids. A) A polynucleotide has a
sugar phosphate backbone with variable appendages the
nitrogenous bases. B) A nucleotide monomer includes a
nitrogenous base, a sugar and a phosphate group.
Without the phosphate group the structure is called a
nucleoside. C) A nucleoside includes a nitrogenous base
(purine or pyrimidine) and a five carbon sugar
deoxyribose or ribose
Deoxyribose (in DNA)
Ribose (in RNA)
(c) Nucleoside components: sugars
Nucleotide Monomers
There are two families of nitrogenous bases:
Pyrimidines (cytosine, thymine, and uracil) have a
single six-membered ring
Purines (adenine and guanine) have a six-membered
ring fused to a five-membered ring
• In DNA, the sugar is deoxyribose; in RNA, the
sugar is ribose
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Nucleotide Polymers
Nucleotide polymers are linked together to build a polynucleotide
Adjacent nucleotides are joined by covalent bonds that form between
the –OH group on the 3′ carbon of one nucleotide and the phosphate
on the 5′ carbon on the next
These links create a backbone of sugar-phosphate units with
nitrogenous bases as appendages.
Covalent bonds in backbone
The sequence of bases along a DNA or mRNA polymer is unique for
each gene
The DNA Double Helix
A DNA molecule has two polynucleotides spiraling around
an imaginary axis, forming a double helix
In the DNA double helix, the two backbones run in opposite
5′ → 3′ directions from each other, an arrangement referred
to as antiparallel
One DNA molecule includes many genes
The nitrogenous bases in DNA pair up and form hydrogen
bonds: adenine (A) always with thymine (T), and guanine
(G) always with cytosine (C)
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RNA
Usually single stranded
Four types of nucleotides
Unlike DNA, contains the
base uracil in place of
thymine
There are several types
of RNA among them are:
mRNA, rRNA and tRNA
which are key players in
protein synthesis
Structure of Nucleotides
in DNA
Each nucleotide consists of
Deoxyribose (5-carbon sugar)
Phosphate group
A nitrogen-containing base
Four bases
Adenine, Guanine, Thymine, Cytosine
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Sugar–phosphate
backbone
5′′ end
The structure of a DNA strand.
Each nucleotide consists of a
nitrogenous base (T, A, C or G), the
sugar deoxyribose (blue) and a
phosphate group (yellow). The
phosphate of one nucleotide is
attached to the sugar of the next
resulting in a backbone of
alternating phosphates and sugars
from which the bases project. The
polynucleotide strand has
directionality from the 5’ end (with
the phosphate group) to the 3’end
(with the –OH group). 5’ and 3’
refer to the numbers assigned to
the carbons in the sugar ring.
Nitrogenous
bases
Thymine (T)
Adenine (A)
Cytosine (C)
Phosphate
DNA nucleotide
Sugar (deoxyribose)
3′′ end
Guanine (G)
5′′ end
Hydrogen bond
3′′ end
1 nm
3.4 nm
3′′ end
0.34 nm
(a) Key features of DNA structure (b) Partial chemical structure
5′′ end
(c) Space-filling model
The double helix. A) The ribbons in this diagram represent the sugar phosphate backbones of
the two DNA strands. The helix is “right handed” curving up to the right. The two strands are
held together by hydrogen bonds (dotted lines) between the nitrogenous bases which are
paired in the interior of the double helix. B) For clarity, the two strands of DNA are shown
untwisted in this partial chemical structure. Strong covalent bonds link the units of each strand,
while weaker hydrogen bonds hold one strand to the other. Notice that the strands are
antiparallel, meaning that they are oriented in opposite directions. C) The tight stacking of the
base pairs is clear in this computer model. Van der Waals attractions between the stacked
pairs play a major role in holding the molecule together.
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Purine + purine: too wide
Pyrimidine + pyrimidine: too narrow
Purine + pyrimidine: width
consistent with X-ray data
Watson-Crick Model
DNA consists of two nucleotide strands
Strands run in opposite directions
Strands are held together by hydrogen bonds between bases
A binds with T and C with G
Molecule is a double helix
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Watson-Crick Model
2-nanometer diameter overall
0.34-nanometer distance between each pair of bases
3.4-nanometer length of each full twist of the double helix
In all respects shown here, the Watson–Crick
model for DNA structure is consistent with the
known biochemical and x-ray diffraction data.
The pattern of base pairing (A only with T, and G only
with C) is consistent with the known composition of
DNA (A = T, and G = C).
Adenine (A)
Thymine (T)
Guanine (G)
Cytosine (C)
Base paring in DNA.
The pairs of nitrogenous
bases in DNA double
helix are held together
by hydrogen bonds
shown here as pink
dotted lines
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Many proteins work together in DNA
replication and repair
DNA is two nucleotide strands held together by hydrogen bonds
Hydrogen bonds between two strands are easily broken
The relationship between structure and function is manifest in the double helix
Watson and Crick noted that the specific base pairing suggested a possible
copying mechanism for genetic material
Each single strand then serves as template for new strand
The Basic Principle: Base Pairing to a
Template Strand
Since the two strands of DNA are complementary,
each strand acts as a template for building a new
strand in replication
In DNA replication, the parent molecule unwinds,
and two new daughter strands are built based on
base-pairing rules
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A
T
A
T
A
T
A
T
C
G
C
G
C
G
C
G
T
A
T
A
T
A
T
A
A
T
A
T
A
T
A
T
G
C
G
C
G
C
G
C
a) The parent molecule has
two complementary
strands of DNA. Each base
is paired by hydrogen
bonding with its specific
partner, A with T and G
with C.
b) The first step in replication is separation of the two DNA
strands. Each parental strand now serves as a template that
determines the order of nucleotides along a new,
complementary strand.
c) The complementary
nucleotides line up and are
connected to form the
sugar-phosphate backbones of the new strands.
Each “daughter” DNA
molecule consists of one
parental strand (dark blue)
and one new strand (light
blue).
A model for DNA replication: the basic concept. In this simplified illustration
a short segment of DNA has been untwisted into a structure that resembles
a ladder. The rolls of the ladder are the sugar phosphate backbones of the
two DNA strands. The rungs are the pairs of nitrogenous bases. Simple
shape symbolizes the four kinds of bases. Dark blue represents DNA
strands present in the parental molecule; light blue represents newly
synthesized DNA.
Watson and Crick’s semiconservative model of
replication predicts that when a double helix
replicates, each daughter molecule will have one
old strand (derived or “conserved” from the parent
molecule) and one newly made strand
Competing models were the conservative model
(the two parent strands rejoin) and the dispersive
model (each strand is a mix of old and new)
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Parent cell
Three alternative
models of DNA
replication. Each
short segment of
double helix
symbolizes the DNA
within a cell.
Beginning with a
parent cell we follow
the DNA for two
generations of cellstwo rounds of DNA
replication. Newly
made DNA is light
blue.
a) Conservative
model. The two
parental strands
reassociate after
acting as templates
for new strands, thus
restoring the parental
double helix.
First
replication
Second
replication
b) Semiconservative
model. The two
strands of the
parental
molecule
separate, and each
functions as a
template for synthesis
of a new,
complementary
strand.
c) Dispersive model.
Each strand of both
daughter molecules
contains
a mixture of
old and newly
synthesized
DNA.
DNA Replication
Replication begins at special sites called origins
of replication, where the two DNA strands are
separated, opening up a replication “bubble”
A eukaryotic chromosome may have hundreds or
even thousands of origins of replication
Replication proceeds in both directions from each
origin, until the entire molecule is copied
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Origin of
replication
Parental (template) strand
Daughter (new) strand
Doublestranded
DNA molecule
Replication fork
Replication
bubble
0.5 µm
Two
daughter
DNA
molecules
(a) Origins of replication in E. coli
In the circular chromosome of E coli and many other bacteria only one origin of replication is
present. The parental strands separate at the origin, forming a replication bubble with two
forks. Replication proceeds in both directions until the forks meet on the other side resulting
in two daughter DNA molecules. The TEM shows a bacterial chromosome with a replication
bubble.
Parental (template) strand
Origin of replication
Bubble
Daughter (new) strand
0.25 µm
Replication fork
Two daughter DNA molecules
a) In each linear chromosomes of eukaryotes, DNA
replication begins when replication bubbles form at
many sites
along the giant DNA molecule of each chromosome.
The bubbles expand as replication proceeds in both
directions. Eventually the bubbles fuse and synthesis
of the daughter strands is complete.
b) This micrograph, shows
three replication
bubbles along the DNA
of a cultured Chinese
hamster cell
(TEM).
Origins of replication in E. coli and eukaryotes. The red arrows indicate the movement
of the replication forks and thus the overall directions of DNA replication within each
bubble
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At the end of each replication bubble is a replication fork,
a Y-shaped region where new DNA strands are elongating
Helicases are enzymes that untwist the double helix at the
replication forks
Single-strand binding protein binds to and stabilizes
single-stranded DNA until it can be used as a template
Topoisomerase corrects “overwinding” ahead of
replication forks by breaking, swiveling, and rejoining DNA
strands
Single-strand binding proteins
stabilize the unwound parental
strands
Primase synthesizes RNA
primers using the parental
DNA as a template
Topoisomerase. Breaks swivels and
rejoins the parental DNA ahead of
the replication fork relieving the
strain caused by unwinding
3′′
5′′
3′′
5′′
RNA
primer
3′′
5′′
Helicase unwinds and separates the
parental DNA strands
Some of the proteins involved in the initiation of DNA replication. The same
proteins function at both replication forks in a replication bubble. For simplicity
only one fork is shown
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DNA polymerases cannot initiate synthesis of a
polynucleotide; they can only add nucleotides to the 3′ end
The initial nucleotide strand is a short RNA primer
An enzyme called primase can start an RNA chain from
scratch and adds RNA nucleotides one at a time using the
parental DNA as a template
The primer is short (5–10 nucleotides long), and the 3′ end
serves as the starting point for the new DNA strand
Synthesizing a New DNA Strand
Enzymes called DNA polymerases catalyze the
elongation of new DNA at a replication fork
Most DNA polymerases require a primer and a DNA
template strand
Each nucleotide that is added to a growing DNA strand is a
nucleoside triphosphate
The rate of elongation is about 500 nucleotides per second
in bacteria and 50 per second in human cells
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Primase synthesizes an RNA primer at the 5′ ends of the
leading strand and the Okazaki fragments
DNA pol III continuously synthesizes the leading strand
and elongates Okazaki fragments
DNA pol I removes primer from the 5′ ends of the leading
strand and Okazaki fragments, replacing primer with DNA
and adding to adjacent 3′ ends
DNA ligase joins the 3′ end of the DNA that replaces the
primer to the rest of the leading strand and also joins the
lagging strand fragments
The DNA Replication Complex
• The proteins that participate in DNA replication form a large
complex, a “DNA replication machine”
• The DNA replication machine is probably stationary during
the replication process
• Recent studies support a model in which DNA polymerase
molecules “reel in” parental DNA and “extrude” newly made
daughter DNA molecules
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New strand
5′′ end
Template strand
3′′ end
5′′ end
3′′ end
Sugar
Base
Phosphate
DNA polymerase
3′′ end
3′′ end
Pyrophosphate
Nucleoside
triphosphate
5′′ end
5′′ end
Incorporation of a nucleotide into a DNA strand. DNA polymerase catalyzes the
addition of a nucleoside triphosphate to the 3’ end of a growing DNA strand with the
release of two phosphates
Each nucleotide that is added to a growing DNA strand is a
nucleoside triphosphate
dATP supplies adenine to DNA and is similar to the ATP of
energy metabolism
The difference is in their sugars: dATP has deoxyribose
while ATP has ribose
As each monomer of dATP joins the DNA strand, it loses
two phosphate groups as a molecule of pyrophosphate
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Antiparallel Elongation
The antiparallel structure of the double helix (two strands
oriented in opposite directions) affects replication
DNA polymerases add nucleotides only to the free 3′ end
of a growing strand; therefore, a new DNA strand can
elongate only in the 5′ to 3′ direction
Along one template strand of DNA, called the leading
strand, DNA polymerase can synthesize a complementary
strand continuously, moving toward the replication fork
To elongate the other new strand, called the lagging
strand, DNA polymerase must work in the direction away
from the replication fork
The lagging strand is synthesized as a series of segments
called Okazaki fragments, which are joined together by
DNA ligase
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Synthesis of the
leading strand during
DNA replication. This
diagram focuses on
the left replication fork
shown in the overview
box. DNA polymerase
III (DNA pol III)
shaped like a cupped
hand is closely
associated with a
protein called the
“sliding clamp” that
encircles the newly
synthesized double
helix like a doughnut.
The sliding clamp
moves DNA pol III
along the DNA
template strand.
Overview
Origin of replication
Leading strand
Lagging strand
Primer
Lagging strand
Leading strand
Overall directions
of replication
Origin of replication
1) After RNA primer is
made DNA pol III starts to
synthesize the leading
strand.
3′′
5′′
RNA primer
5′′
“Sliding clamp”
3′′
5′′
Parental DNA
DNA poll III
3′′
5′′
5′′
3′′
5′′
2) The leading strand is
elongated continuously in
the 5’ →3’ direction as the
fork progresses
Strand Assembly
Why the discontinuous
additions? Nucleotides can
only be joined to an exposed —
OH group that is attached to
the 3’ carbon of a growing
strand. This is why we say that
DNA and RNA synthesis occurs
in the 5’ to 3’ direction
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Continuous and
Discontinuous Assembly
As Reiji Okazaki discovered, strand
assembly is continuous on just one
parent strand. This is because DNA
synthesis occurs only in the 5´ to 3´
direction. On the other strand,
assembly is discontinuous: short,
separate stretches of nucleotides
are added to the template, and then
enzymes fill in the gaps between
them.
Continuous and Discontinuous
Assembly
Strands can only
be assembled in
the 5’ to 3’
direction
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Overview
Origin of replication
Leading strand
Lagging strand
Lagging strand
2
1
Leading strand
Overall directions
of replication
3′′
5′′
5′′
3′′
Template
strand
3′′
RNA primer
3′′
5′′
5′′
Okazaki
fragment
3′′
3′′
5′′
5′′
Synthesis of
the lagging
strand
1) Primase joins RNA
nucleotides into a primer.
5′′
3′′
3′′
5′′
5′′
3′′
3′′
5′′
5′′
3′′
3′′
5′′
Overall direction of replication
2) DNA pol III adds
DNA nucleotides to
the primer, forming
an Okazaki fragment 1.
3) After reaching the
next RNA primer to the right DNA pol III
detaches .
4) After fragment 2 is
primed, DNA pol III adds DNA
nucleotides until it reaches fragment 1
and detaches.
5) DNA pol I replaces
the RNA with DNA,
adding to the 3′′ end
of fragment 2.
6) DNA ligase forms a
bond between the newest
DNA and the adjacent DNA
of fragment 1.
7) The lagging
strand in this region
is now complete.
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Overview
Origin of replication
Lagging strand
Leading strand
1) Helicase
unwinds
the parental
double
helix
2) Molecules of singlestrand binding protein
stabilize the unwound
template strands
Leading strand
Lagging strand
Overall directions
of replication
3) The leading strand is
synthesized continuously in the 5
→ 3 direction by DNA pol III.
DNA pol III
4)
Primase
begins
synthesis
of the RNA
3′′
7) DNA ligase
Primer
primer for the fifth Okazaki fragment
bonds the 3 ‘ end of
5′′
3′′
Parental DNA
the second
Lagging strand
DNA pol III
fragment to the 5’
5′′
end of the first
DNA
pol
I
4
3′′ 5′′
5) DNA pol III is completing
fragment
3
synthesis of the fourth fragment.
2
1
3′′
When it reaches the RNA
5′′
primer on the third fragment it
will dissociate move to the
6) DNA pol I removes the primer from the 5’ end of the second fragment
replication fork and add DNA
and replaces it with DNA nucleotides that it adds one by one to the 3’ end
nucleotides to the 3’ end of the
of the third fragment. The replacement of the last RNA nucleotide with
fifth fragment primer
DNA leaves the sugar phosphate backbone with a free 3’ end.
5′′
3′′
A summary of bacterial DNA replication. The detailed diagram shows one replication fork, but as indicated
in the overview (upper right) replication usually occurs simultaneously at two forks one at either end of the
replication bubble. Viewing each daughter strand in its entirety in the overview, you can see that half of it is
made continuously as the leading strand while the other half (on the other side of the origin) is synthesized
in fragments as the lagging strand.
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Proofreading and Repairing DNA
Mistakes can occur during replication
DNA polymerase can read correct sequence from complementary strand
and, together with DNA ligase, can repair mistakes in incorrect strandproofreading activity
DNA polymerases proofread newly made DNA, replacing any incorrect
nucleotides
In mismatch repair of DNA, repair enzymes correct errors in base pairing
DNA can be damaged by chemicals, radioactive emissions, X-rays, UV
light, and certain molecules (in cigarette smoke for example)
In nucleotide excision repair, a nuclease cuts out and replaces
damaged stretches of DNA
A thymine dimer
distorts the DNA molecule.
Nucleotide excision repair of
DNA damage. A team of
enzymes detects and repairs
damaged DNA. This figure
shows DNA containing a
thymine dimer, a type of
damage often caused by
ultraviolet radiation. A nuclease
enzyme cuts out the damaged
region of DNA and a DNA
polymerase (in bacteria DNA
pol I) replaces it with
nucleotides complementary to
the undamaged strand. DNA
ligase completes the process
by closing the remaining break
in the sugar phosphate
backbone.
A nuclease enzyme cuts
the damaged DNA strand
at two points and the
damaged section is
removed.
Nuclease
Repair synthesis by
a DNA polymerase
fills in the missing
nucleotides.
DNA
polymerase
DNA
ligase
DNA ligase seals the
free end of the new DNA
to the old DNA, making the
strand complete.
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Replicating the Ends of DNA
Molecules
Limitations of DNA polymerase create problems for the
linear DNA of eukaryotic chromosomes
The usual replication machinery provides no way to
complete the 5′ ends, so repeated rounds of replication
produce shorter DNA molecules
5′′
Leading strand
Lagging strand
End of parental
DNA strands
3′′
Shortening of the ends of
linear DNA molecules. Here
we follow the end of one
strand of a DNA molecule
through two rounds of
replication. After the first
round the new lagging
strand is shorter than its
template. After a second
round both the leading and
lagging strands have
become shorter than the
original parental DNA.
Although not shown here
the other ends of these
DNA molecules also
become shorter.
Last fragment
Previous fragment
RNA primer
Lagging strand 5′′
3′′
Primer removed but
cannot be replaced
with DNA because
no 3′′ end available
for DNA polymerase
Removal of primers and
replacement with DNA
where a 3′′ end is available
5′′
3′′
Second round
of replication
5′′
New leading strand 3′′
New leading strand 5′′
3′′
Further rounds
of replication
Shorter and shorter
daughter molecules
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Eukaryotic chromosomal DNA molecules have at their ends nucleotide
sequences called telomeres
Telomeres do not prevent the shortening of DNA molecules, but they
do postpone the erosion of genes near the ends of DNA molecule.
Telomeres are involved in protecting chromosomes
It has been proposed that the shortening of telomeres is connected to
aging. Since telomeric sequences shorten each time the DNA
replicates telomeres are also thought to be a molecular "clock" that
regulates how many times an individual cell can divide. After a certain
number of divisions the telomere shrinks to a certain level thereby
causing the cell to stop dividing.The cell’s metabolism slows down, it
ages, and dies.
Telomeres
Telomeres exist at the ends of a chromosome
They contain multiple copies of a G rich sequence
In humans this sequence is TTAGGG, which can be repeated
several thousand times
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If chromosomes of germ cells became shorter in every cell
cycle, essential genes would eventually be missing from the
gametes they produce
An enzyme called telomerase catalyzes the lengthening of
telomeres in germ cells
The shortening of telomeres might protect cells from
cancerous growth by limiting the number of cell divisions
There is evidence of telomerase activity in cancer cells,
which may allow cancer cells to persist
Telomerase
Shay et al. found that cellular aging can be bypassed or put on hold
by the addition of the enzymatic component of telomerase
Telomerase is a ribonucleoprotein enzyme complex. It consists of
two components
Protein- enzyme known as reverse transcriptase (RT)
RNA- serves as the template
The RNA functions as a template for the reverse transcriptase. The
RT as the name suggests uses RNA as template to make DNA. So
in this case the RT adds nucleotides to the chromosomal endstelomeres- thereby extending them.
It stabilizes telomere length by adding hexameric (TTAGGG)
repeats onto the telomeric ends of the chromosomes
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Telomerase function
Normal cells undergo a certain number of cell divisions and then
seize to divide.
Most normal cells do not have this enzyme and thus they lose
telomeres with each division.
If these cells are grown in tissue culture with the enzyme
telomerase
Their telomeres get extended
They can continue to divide hundreds of generations past the
time they normally would stop dividing.
In humans, telomerase is active in germ cells, in the vast majority of
cancer cells and, possibly, in some stem cells, epidermal skin cells
and follicular hair cells.
A chromosome consists of a DNA
molecule packed together with proteins
The bacterial chromosome is a double-stranded,
circular DNA molecule associated with a small amount
of protein
Eukaryotic chromosomes have linear DNA molecules
associated with a large amount of protein
In a bacterium, the DNA is “supercoiled” and found in
a region of the cell called the nucleoid
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Chromatin is a complex of DNA and protein, and
is found in the nucleus of eukaryotic cells
Histones are proteins that are responsible for the
first level of DNA packing in chromatin
Chromatin packing in a eukaryotic chromosome
This series of diagrams and transmission electron micrographs depicts a
current model for the progressive levels of DNA coiling and folding. The
illustration zooms out from a single molecule of DNA to a metaphase
chromosome, which is large
enough to be seen with a light
microscope.
DNA
helix
nm in diameter)
Nucleosome
(10 nm in diameter)
double
(2
H1
Histones
1) DNA, the double helix
Shown here is a ribbon
model of DNA, with
each ribbon
representing one of the
sugar-phosphate
backbones. As you will
recall from Figure 16.7,
the phosphate groups
along the backbone
contribute a negative
charge along the
outside of each strand.
The TEM shows a
molecule of naked DNA;
the double helix alone is
2 nm across.
Histone tail
2) Histones. Proteins called histones are
responsible for the first level of DNA packing in
chromatin. Although each histone is small—
containing about 100 amino acids—the total mass
of histone in chromatin approximately equals the
mass of DNA. More than a fifth of a histone's amino
acids are positively charged (lysine or arginine) and
bind tightly to the negatively charged DNA.
Four types of histones are most common in
chromatin: H2A, H2B, H3, and H4. The histones
are very similar among eukaryotes; for example, all
but two of the amino acids in cow H4 are identical
to those in pea H4. The apparent conservation of
histone genes during evolution probably reflects the
pivotal role of histones in organizing DNA within
cells.
The four main types of histones are critical to the
next level of DNA packing. (A fifth type of histone,
called H1, is involved in a further stage of packing)
Histones
3) Nucleosomes, or “beads on a string” (10-nm
fiber). In electron micrographs, unfolded
chromatin is 10 nm in diameter (the 10-nm fiber).
Such chromatin resembles beads on a string Each "bead" is a nucleosome the basic unit of
DNA packing; the "string" between beads is
called linker DNA.
A nucleosome consists of DNA wound twice
around a protein core composed of two
molecules each of the four main histone types.
The amino end (N-terminus) of each histone (the
histone tail) extends outward from the
nucleosome.
In the cell cycle, the histones leave the DNA only
briefly during DNA replication. Generally they do
the same during gene expression, another
process that requires access to the DNA by the
cell's molecular machinery. Chapter 18 will
discuss some recent findings about the role of
histone tails and nucleosomes in gene regulation
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Chromatin packing in a eukaryotic chromosome
Chromatid
(700 nm)
30-nm fiber
Loops
Scaffold
300-nm fiber
Replicated chromosome
(1,400 nm)
4) 30-nm fiber. The next level of packing is due
to interactions between the histone tails of one
nucleosome and he linker DNA and nudeosomes
on either side. A fifth histone, H1, is involved at
this level. These interactions cause the extended
10-nm fiber to coil or fold, forming a chromatin
fiber roughly 30 nm in thickness, the 30-nm fiber.
Although the 30-nm fiber is quite prevalent in
e interphase nucleus, the packing arrangement
of nudeosomes in this form of chromatin is still a
matter of some debate
5) Looped domains (300-nm
fiber)
The 30-nm fiber, in turn, forms
loops called looped domains
attached to a chromosome
scaffold made of proteins, thus
making up a 300-nm fiber. The
scaffold is rich in one type of
topoisomerase, and Hl molecules
also appear to be present
6) Metaphase chromosome In a mitotic chromosome,
the looped domains themselves coil and fold in a manner
not yet fully understood, further compacting all the
chromatin to produce the characteristic metaphase
chromosome shown in the micrograph above. The width
of one chromatid is 700 nm. Particular genes always end
up located at the same places in metaphase
chromosomes, indicating that the packing steps are highly
specific and precise
Chromatin is organized into fibers
10-nm fiber
DNA winds around histones to form nucleosome “beads”
Nucleosomes are strung together like beads on a string by linker DNA
30-nm fiber
Interactions between nucleosomes cause the thin fiber to coil or fold into
this thicker fiber
300-nm fiber
The 30-nm fiber forms looped domains that attach to proteins
Metaphase chromosome
The looped domains coil further
The width of a chromatid is 700 nm
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Most chromatin is loosely packed in the nucleus during interphase
and condenses prior to mitosis
Loosely packed chromatin is called euchromatin
During interphase a few regions of chromatin (centromeres and
telomeres) are highly condensed into heterochromatin
Dense packing of the heterochromatin makes it difficult for the cell to
express genetic information coded in these regions
Histones can undergo chemical modifications that result in changes
in chromatin organization
For example, phosphorylation of a specific amino acid on a
histone tail affects chromosomal behavior during meiosis
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