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Biology Review

� Cell Structure

� Biological Molecules (DNA & Proteins)

� Central Dogma

� Structure and Function of DNA

� Replication, Transcription, Translation

� Regulation of Gene Expression

1 µmOrganelles

Nucleus (contains DNA)

Cytoplasm

Membrane

DNA(no nucleus)

Membrane

Eukaryotic cell

Prokaryotic cell

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Fimbriae

Capsule

Cell wall

Circular chromosome

Internalorganization

Flagella

Sex pilus

Prokaryotic cell

ENDOPLASMIC RETICULUM (ER)

Smooth ERRough ERFlagellum

Centrosome

CYTOSKELETON:

Microfilaments

Intermediatefilaments

Microtubules

Microvilli

Peroxisome

MitochondrionLysosome

Golgiapparatus

Ribosomes

Plasma membrane

Nuclearenvelope

Nucleolus

Chromatin

NUCLEUS

Animal cell

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Cell Component Structure Function

The eukaryotic cell’s geneticinstructions are housed inthe nucleus and carried outby the ribosomes

Nucleus Surrounded by nuclearenvelope (double membrane)perforated by nuclear pores.The nuclear envelope iscontinuous with theendoplasmic reticulum (ER).

(ER)

Houses chromosomes, made ofchromatin (DNA, the geneticmaterial, and proteins); containsnucleoli, where ribosomalsubunits are made. Poresregulate entry and exit osmaterials.

Ribosome Two subunits made of ribo-somal RNA and proteins; can befree in cytosol or bound to ER

Protein synthesis

Cell Component Structure Function

The endomembrane systemregulates protein traffic andperforms metabolic functionsin the cell

Endoplasmic reticulum

(Nuclearenvelope)

Golgi apparatus

Lysosome

Vacuole Large membrane-boundedvesicle in plants

Membranous sac of hydrolyticenzymes (in animal cells)

Stacks of flattenedmembranoussacs; has polarity(cis and trans

faces)

Extensive network ofmembrane-bound tubules andsacs; membrane separateslumen from cytosol;continuous withthe nuclear envelope.

Smooth ER: synthesis oflipids, metabolism of carbohy-drates, Ca2+ storage, detoxifica-tion of drugs and poisons

Rough ER: Aids in sythesis ofsecretory and other proteinsfrom bound ribosomes; addscarbohydrates to glycoproteins;produces new membrane

Modification of proteins, carbo-hydrates on proteins, and phos-pholipids; synthesis of manypolysaccharides; sorting ofGolgi products, which are thenreleased in vesicles.

Breakdown of ingested sub-stances cell macromolecules, and damaged organelles for recycling

Digestion, storage, wastedisposal, water balance, cellgrowth, and protection

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Cell Component

Mitochondria and chloro-plasts change energy fromone form to another

Mitochondrion

Chloroplast

Peroxisome

Structure Function

Bounded by doublemembrane;inner membrane hasinfoldings (cristae)

Typically two membranesaround fluid stroma, whichcontains membranous thylakoidsstacked into grana (in plants)

Specialized metaboliccompartment bounded by asingle membrane

Cellular respiration

Photosynthesis

Contains enzymes that transferhydrogen to water, producinghydrogen peroxide (H2O2) as aby-product, which is convertedto water by other enzymesin 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

Aminogroup

Carboxylgroup

αααα carbon

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Nonpolar

Glycine(Gly or G)

Alanine(Ala or A)

Valine(Val or V)

Leucine(Leu or L)

Isoleucine(Ile or ΙΙΙΙ)

Methionine(Met or M)

Phenylalanine(Phe or F)

Trypotphan(Trp or W)

Proline(Pro or P)

Polar

Serine(Ser or S)

Threonine(Thr or T)

Cysteine(Cys or C)

Tyrosine(Tyr or Y)

Asparagine(Asn or N)

Glutamine(Gln or Q)

Electricallycharged

Acidic Basic

Aspartic acid(Asp or D)

Glutamic acid(Glu or E)

Lysine(Lys or K)

Arginine(Arg or R)

Histidine(His or H)

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

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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Peptidebond

Fig. 5-18

Amino end(N-terminus)

Peptidebond

Side chains

Backbone

Carboxyl end(C-terminus)

(a)

(b)

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.

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 three-dimensional structure

�A protein’s structure determines its function

GrooveGroove

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.

PrimaryStructure

SecondaryStructure

TertiaryStructure

β pleated sheet

Examples ofamino acidsubunits

+H3NAmino end

α helix

QuaternaryStructure

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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-

Amino acidsubunits

+H3NAmino end

25

20

15

10

5

1

Primary StructureAmino acidsubunits

+H3NAmino end

Carboxyl end125

120

115

110

105

100

95

9085

80

75

20

25

15

10

5

1

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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 αααα helix and a folded structure called a ββββpleated sheet

Secondary Structure

ββββ pleated sheet

Examples ofamino acidsubunits

αααα helix

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� 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

Tertiary Structure Quaternary Structure

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Polypeptidebackbone

Hydrophobicinteractions andvan der Waalsinteractions

Disulfide bridge

Ionic bond

Hydrogenbond

Polypeptidechain

β β β β −−−− Chains

Heme

Iron

α α α α −−−− Chains

Collagen

Hemoglobin

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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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Normal protein Denatured protein

Denaturation

Renaturation

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

DiffractedX-rays

EXPERIMENT

X-raysource X-ray

beam

Crystal Digital detector X-ray diffractionpattern

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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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mRNA

Synthesis ofmRNA in thenucleus

DNA

NUCLEUS

mRNA

CYTOPLASM

Movement ofmRNA into cytoplasmvia nuclear pore

Ribosome

AminoacidsPolypeptide

Synthesisof protein

1

2

3

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

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

Nucleoside

Nitrogenousbase

Phosphategroup Sugar

(pentose)

(b) Nucleotide

(a) Polynucleotide, or nucleic acid

3 end

3C

3C

5C

5C

Nitrogenous bases

Pyrimidines

Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA)

Purines

Adenine (A) Guanine (G)

Sugars

Deoxyribose (in DNA) Ribose (in RNA)

(c) Nucleoside components: sugars

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

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

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

DNA nucleotide

Sugar (deoxyribose) 3′′′′ end

Phosphate

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.

(c) Space-filling model

Hydrogen bond 3′′′′ end

5′′′′ end

3.4 nm

0.34 nm

3′′′′ end

5′′′′ end

(b) Partial chemical structure(a) Key features of DNA structure

1 nm

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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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).

Watson-Crick Model

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

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

GC

T A

TA

G C

A T

GC

T A

TA

G C

A T

GC

T A

TA

G C

A T

GC

T A

TA

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 back-

bones 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 cellFirst replication

Second replication

a) Conservative model. The two parental strands reassociate after

acting as templates for new strands, thus restoring the parental

double helix.

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.

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 cells-

two rounds of DNA

replication. Newly

made DNA is light

blue.

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

Replication fork

Replication bubble

Double-stranded DNA molecule

Two daughter DNA molecules

(a) Origins of replication in E. coli

0.5 µm

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.

Two daughter DNA molecules

Parental (template) strand

Daughter (new) strand0.25 µm

Replication fork

Origin of replication

Bubble

a) In each linear chromosomes of eukaryotes, DNA replication begins when replication bubbles form at

many sitesalong 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 DNAof 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

RNA primer

5′′′′5′′′′

5′′′′ 3′′′′

3′′′′

3′′′′

Single-strand binding proteins stabilize the unwound parental

strands

Primase synthesizes RNA primers using the parental

DNA as a template

Helicase unwinds and separates the parental DNA strands

Topoisomerase. Breaks swivels and rejoins the parental DNA ahead of the replication fork relieving the

strain caused by unwinding

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

PhosphateBase

Sugar

Template strand

3′′′′ end 5′′′′ end 3′′′′ end

5′′′′ end

3′′′′ end

5′′′′ end

3′′′′ end

Nucleosidetriphosphate

DNA polymerase

Pyrophosphate

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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Leading strand

Overview

Origin of replication

Lagging strand

Leading strandLagging strand

Primer

Overall directions of replication

Origin of replication

RNA primer

“Sliding clamp”

DNA poll IIIParental DNA

5′′′′

3′′′′

3′′′′

3′′′′

3′′′′

5′′′′

5′′′′

5′′′′

5′′′′

5′′′′

1) After RNA primer is

made DNA pol III starts to

synthesize the leading

strand.

2) The leading strand is

elongated continuously in

the 5’ →3’ direction as the

fork progresses

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.

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

Strand AssemblyStrand Assembly

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As Reiji Okazaki discovered, strand assembly is continuous on just one parent strand. This is because DNAsynthesis 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 Continuous and Discontinuous AssemblyDiscontinuous Assembly

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

Leading strand

Lagging strand

Lagging strand

Overall directions of replication

12

5 ′′′′3 ′′′′

Templatestrand

5 ′′′′ 3 ′′′′

Overall direction of replication

RNA primer3 ′′′′

5 ′′′′

3 ′′′′5 ′′′′

Okazakifragment

3 ′′′′

5 ′′′′

5 ′′′′

3 ′′′′

3 ′′′′3 ′′′′

5 ′′′′

5 ′′′′

3 ′′′′3 ′′′′

5 ′′′′

5 ′′′′

3 ′′′′

3 ′′′′

5 ′′′′

5 ′′′′

1) Primase joins RNAnucleotides into a primer.

2) DNA pol III addsDNA nucleotides to the primer, forming

an Okazaki fragment 1.

3) After reaching thenext RNA primer to the right DNA pol III

detaches .

4) After fragment 2 isprimed, 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′′′′ endof fragment 2.

6) DNA ligase forms abond between the newestDNA and the adjacent DNA

of fragment 1.

7) The lagging strand in this region

is now complete.

Synthesis of

the lagging

strand

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OverviewOrigin of replication

Leading strand

Leading strand

Lagging strand

Lagging strandOverall directions

of replication

Lagging strand

1) Helicase unwinds

the parental double helix

Parental DNA

DNA pol III

Primer

DNA pol III

DNA pol I

5′′′′

3′′′′

5′′′′

5′′′′

5′′′′

5′′′′

3′′′′

3′′′′

3′′′′

3′′′′13

2

4

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.

3) The leading strand is

synthesized continuously in the 5

→ 3 direction by DNA pol III.

2) Molecules of single-strand binding protein stabilize the unwound

template strands

4) Primase begins synthesis of the RNA primer for the fifth Okazaki fragment 7) DNA ligase

bonds the 3 ‘ end of

the second

fragment to the 5’

end of the first

fragment5) DNA pol III is completing

synthesis of the fourth fragment.

When it reaches the RNA

primer on the third fragment it

will dissociate move to the

replication fork and add DNA

nucleotides to the 3’ end of the

fifth fragment primer

6) DNA pol I removes the primer from the 5’ end of the second fragment

and replaces it with DNA nucleotides that it adds one by one to the 3’ end

of the third fragment. The replacement of the last RNA nucleotide with

DNA leaves the sugar phosphate backbone with a free 3’ end.

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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 strand-

proofreading 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

DNA ligase

DNA polymerase

DNA ligase seals thefree end of the new DNAto the old DNA, making thestrand complete.

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

A nuclease enzyme cutsthe damaged DNA strandat two points and the damaged section isremoved.

Nuclease

A thymine dimerdistorts 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.

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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

End of parentalDNA strands

5′′′′

3′′′′

Lagging strand 5′′′′

3′′′′

Last fragment

RNA primer

Leading strand

Lagging strand

Previous fragment

Primer removed butcannot be replacedwith DNA becauseno 3′′′′ end available

for DNA polymerase5′′′′

3′′′′

Removal of primers andreplacement with DNAwhere a 3′′′′ end is available

Second roundof replication

5′′′′

3′′′′

5′′′′

3′′′′

Further roundsof replication

New leading strand

New leading strand

Shorter and shorterdaughter molecules

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.

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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 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

Telomeres

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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 ends-

telomeres- 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

DNA double helix (2 nm in diameter)

Nucleosome(10 nm in diameter)

HistonesHistone tail

H1

Histones1) 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.

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)

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

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.

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30-nm fiber

Chromatid (700 nm)

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 ine interphase nucleus, the packing arrangement of nudeosomes in this form of chromatin is still a matter of some debate

Chromatin packing in a eukaryotic chromosome

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