Molecular biology for post graduates.ppt

© 2011 Pearson Education, Inc.
A- The Molecular Basis of Inheritance
Advanced Molecular Biology

Overview: Life’s Operating Instructions
• In 1953, James Watson and Francis Crick
introduced an elegant double-helical model for the
structure of deoxyribonucleic acid, or DNA
• DNA, the substance of inheritance, is the most
celebrated molecule of our time
• Hereditary information is encoded in DNA and
reproduced in all cells of the body
• This DNA program directs the development of
biochemical, anatomical, physiological, and (to
some extent) behavioral traits

DNA is the genetic material
• Early in the 20th century, the identification of the
molecules of inheritance loomed as a major
challenge to biologists

The Search for the Genetic Material:
Scientific Inquiry
• When T. H. Morgan’s group showed that genes
are located on chromosomes, the two components
of chromosomes—DNA and protein—became
candidates for the genetic material
• The key factor in determining the genetic material
was choosing appropriate experimental organisms
• The role of DNA in heredity was first discovered by
studying bacteria and the viruses that infect them

Evidence That DNA Can Transform
Bacteria
• The discovery of the genetic role of DNA began
with research by Frederick Griffith in 1928
• Griffith worked with two strains of a bacterium, one
pathogenic and one harmless

• When he mixed heat-killed remains of the
pathogenic strain with living cells of the harmless
strain, some living cells became pathogenic
• He called this phenomenon transformation, now
defined as a change in genotype and phenotype
due to assimilation of foreign DNA

Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells
(control)
Mixture of
heat-killed
S cells and
living R cells
Mouse dies Mouse dies
Mouse healthy Mouse healthy
Living S cells
EXPERIMENT
RESULTS

• In 1944, Oswald Avery, Maclyn McCarty, and
Colin MacLeod announced that the transforming
substance was DNA
• Their conclusion was based on experimental
evidence that only DNA worked in transforming
harmless bacteria into pathogenic bacteria
• Many biologists remained skeptical, mainly
because little was known about DNA

Evidence That Viral DNA Can Program
Cells
• More evidence for DNA as the genetic material
came from studies of viruses that infect bacteria
• Such viruses, called bacteriophages (or phages),
are widely used in molecular genetics research

Figure 16.3
Phage
head
Tail
sheath
Tail fiber
DNA
Bacterial
cell
100
nm

• In 1952, Alfred Hershey and Martha Chase
performed experiments showing that DNA is the
genetic material of a phage known as T2
• To determine this, they designed an experiment
showing that only one of the two components of
T2 (DNA or protein) enters an E. coli cell during
infection
• They concluded that the injected DNA of the
phage provides the genetic information

Figure 16.4-1
Bacterial cell
Phage
Batch 1:
Radioactive
sulfur
(35S)
DNA
Batch 2:
Radioactive
phosphorus
(32P)
Radioactive
DNA
EXPERIMENT
Radioactive
protein

Figure 16.4-2
Bacterial cell
Phage
Batch 1:
Radioactive
sulfur
(35S)
Radioactive
protein
DNA
Batch 2:
Radioactive
phosphorus
(32P)
Radioactive
DNA
Empty
protein
shell
Phage
DNA
EXPERIMENT

Figure 16.4-3
Bacterial cell
Phage
Batch 1:
Radioactive
sulfur
(35S)
Radioactive
protein
DNA
Batch 2:
Radioactive
phosphorus
(32P)
Radioactive
DNA
Empty
protein
shell
Phage
DNA
Centrifuge
Centrifuge
Radioactivity
(phage protein)
in liquid
Pellet (bacterial
cells and contents)
Pellet
Radioactivity
(phage DNA)
in pellet
EXPERIMENT

Additional Evidence That DNA Is the
Genetic Material
• It was known that DNA is a polymer of nucleotides,
each consisting of a nitrogenous base, a sugar,
and a phosphate group
• In 1950, Erwin Chargaff reported that DNA
composition varies from one species to the next
• This evidence of diversity made DNA a more
credible candidate for the genetic material

• Two findings became known as Chargaff’s rules
– The base composition of DNA varies between
species
– In any species the number of A and T bases are
equal and the number of G and C bases are equal
• The basis for these rules was not understood until
the discovery of the double helix

Figure 16.5
Sugar–phosphate
backbone
Nitrogenous bases
Thymine (T)
Adenine (A)
Cytosine (C)
Guanine (G)
Nitrogenous base
Phosphate
DNA
nucleotide
Sugar
(deoxyribose)
3 end
5 end

Building a Structural Model of DNA:
Scientific Inquiry
• After DNA was accepted as the genetic material,
the challenge was to determine how its structure
accounts for its role in heredity
• Maurice Wilkins and Rosalind Franklin were using
a technique called X-ray crystallography to study
molecular structure
• Franklin produced a picture of the DNA molecule
using this technique

Figure 16.6b
(b) Franklin’s X-ray diffraction
photograph of DNA

• Franklin’s X-ray crystallographic images of DNA
enabled Watson to deduce that DNA was helical
• The X-ray images also enabled Watson to deduce
the width of the helix and the spacing of the
nitrogenous bases
• The pattern in the photo suggested that the DNA
molecule was made up of two strands, forming a
double helix

Figure 16.7
3.4 nm
1 nm
0.34 nm
Hydrogen bond
(a) Key features of
DNA structure
Space-filling
model
(c)
(b) Partial chemical structure
3 end
5 end
3 end
5 end
T
T
A
A
G
G
C
C
C
C
C
C
C
C
C
C
C
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
A
A
A
A
A
A

3.4 nm
1 nm
0.34 nm
Hydrogen bond
(a) Key features of
DNA structure
(b) Partial chemical structure
3 end
5 end
3 end
5 end
T
T
A
A
G
G
C
C
C
C
C
C
C
C
C
C
C
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
A
A
A
A
A
A
Figure 16.7a

Figure 16.7b
(c) Space-filling model

• Watson and Crick built models of a double helix to
conform to the X-rays and chemistry of DNA
• Franklin had concluded that there were two outer
sugar-phosphate backbones, with the nitrogenous
bases paired in the molecule’s interior
• Watson built a model in which the backbones were
antiparallel (their subunits run in opposite
directions)

• At first, Watson and Crick thought the bases paired
like with like (A with A, and so on), but such
pairings did not result in a uniform width
• Instead, pairing a purine with a pyrimidine resulted
in a uniform width consistent with the X-ray data

Figure 16.UN01
Purine  purine: too wide
Pyrimidine  pyrimidine: too narrow
Purine  pyrimidine: width
consistent with X-ray data

• Watson and Crick reasoned that the pairing was
more specific, dictated by the base structures
• They determined that adenine (A) paired only with
thymine (T), and guanine (G) paired only with
cytosine (C)
• The Watson-Crick model explains Chargaff’s
rules: in any organism the amount of A = T, and
the amount of G = C

Figure 16.8
Sugar
Sugar
Sugar
Sugar
Adenine (A) Thymine (T)
Guanine (G) Cytosine (C)

Many proteins work together in DNA
replication and repair
• 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

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

Figure 16.9-1
(a) Parent molecule
A
A
A
T
T
T
C
C
G
G

Figure 16.9-2
(a) Parent molecule (b) Separation of
strands
A
A
A
A
A
A
T
T
T
T
T
T
C
C
C
C
G
G
G
G

Figure 16.9-3
(a) Parent molecule (b) Separation of
strands
(c)“Daughter” DNA molecules,
each consisting of one
parental strand and one
new strand
A
A
A
A
A
A
A
A
A
A
A
A
T
T
T
T
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
G
G
G
G
G
G
G
G

• 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)

Figure 16.10
(a) Conservative
model
(b) Semiconservative
model
(c) Dispersive model
Parent
cell
First
replication
Second
replication

• Experiments by Matthew Meselson and Franklin
Stahl supported the semiconservative model
• They labeled the nucleotides of the old strands
with a heavy isotope of nitrogen, while any new
nucleotides were labeled with a lighter isotope

• The first replication produced a band of hybrid
DNA, eliminating the conservative model
• A second replication produced both light and
hybrid DNA, eliminating the dispersive model and
supporting the semiconservative model

Figure 16.11
Bacteria
cultured in
medium with
15N (heavy
isotope)
Bacteria
transferred to
medium with
14N (lighter
isotope)
DNA sample
centrifuged
after first
replication
DNA sample
centrifuged
after second
replication
Less
dense
More
dense
Predictions: First replication Second replication
Conservative
model
Semiconservative
model
Dispersive
model
2
1
3 4
EXPERIMENT
RESULTS
CONCLUSION

Figure 16.11a
Bacteria
cultured in
medium with
15N (heavy
isotope)
Bacteria
transferred to
medium with
14N (lighter
isotope)
DNA sample
centrifuged
after first
replication
DNA sample
centrifuged
after second
replication
Less
dense
More
dense
2
1
3 4
EXPERIMENT
RESULTS

Figure 16.11b
Predictions: First replication Second replication
Conservative
model
Semiconservative
model
Dispersive
model
CONCLUSION

DNA Replication: A Closer Look
• The copying of DNA is remarkable in its speed
and accuracy
• More than a dozen enzymes and other proteins
participate in DNA replication

Getting Started
• Replication begins at particular 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

Figure 16.12
(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell
Origin of
replication
Parental (template) strand
Double-
stranded
DNA molecule
Daughter (new)
strand
Replication
fork
Replication
bubble
Two daughter
DNA molecules
Origin of replication
Double-stranded
DNA molecule
Parental (template)
strand
Daughter (new)
strand
Bubble Replication fork
Two daughter DNA molecules
0.5
m
0.25
m

Figure 16.12a
(a) Origin of replication in an E. coli cell
Origin of
replication Parental (template) strand
Double-
stranded
DNA molecule
Daughter (new) strand
Replication fork
Replication
bubble
Two
daughter
DNA molecules
0.5 m

Figure 16.12b
(b) Origins of replication in a eukaryotic cell
Double-stranded
DNA molecule
Parental (template)
strand
Daughter (new)
strand
Bubble Replication fork
Two daughter DNA molecules
0.25 m

• 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 proteins bind to and
stabilize single-stranded DNA
• Topoisomerase corrects “overwinding” ahead of
replication forks by breaking, swiveling, and
rejoining DNA strands

Figure 16.13
Topoisomerase
Primase
RNA
primer
Helicase
Single-strand binding
proteins
5
3
5
5
3
3

• 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
• The rate of elongation is about 500 nucleotides
per second in bacteria and 50 per second in
human cells

• 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

Figure 16.14
New strand Template strand
Sugar
Phosphate Base
Nucleoside
triphosphate
DNA
polymerase
Pyrophosphate
5
5
5
5
3
3
3
3
OH
OH
P P i
2 P i
A
A
A
A
T T
T
C
C
C
C
C
C
G
G
G
G

Antiparallel Elongation
• The antiparallel structure of the double helix
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, the DNA
polymerase synthesizes a leading strand
continuously, moving toward the replication fork

Figure 16.15
Leading
strand
Lagging
strand
Overview
Origin of replication Lagging
strand
Leading
strand
Primer
Overall directions
of replication
Origin of
replication
RNA primer
Sliding clamp
DNA pol III
Parental DNA
3
5
5
3
3
5
3
5
3
5
3
5

Figure 16.15a
Leading
strand
Lagging
strand
Overview
Origin of replication Lagging
strand
Leading
strand
Primer
Overall directions
of replication

Origin of
replication
RNA primer
Sliding clamp
DNA pol III
Parental DNA
3
5
5
3
3
5
3
5
3
5
3
5
Figure 16.15b

• 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

Overview
Leading
strand
Leading
strand
Lagging
strand
Lagging strand
Overall directions
of replication
Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
RNA primer
for fragment 2
Okazaki
fragment 2
Overall direction of replication
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
5
5
5
5
2
2
2
1
1
1
1
1
2
1
Figure 16.16

Figure 16.16a
Overview
Leading
strand
Leading
strand
Lagging
strand
Lagging strand
Overall directions
of replication
1
2

Figure 16.16b-1
Template
strand
3
3
5
5

Figure 16.16b-2
Template
strand
RNA primer
for fragment 1
3
3
3
3
5
5
5
5
1

Figure 16.16b-3
Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
3
3
3
3
3
3
5
5
5
5
5
5
1
1

Figure 16.16b-4
Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
RNA primer
for fragment 2
Okazaki
fragment 2
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
2
1
1
1

Figure 16.16b-5
Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
RNA primer
for fragment 2
Okazaki
fragment 2
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
5
5
5
2
2
1
1
1
1

Figure 16.16b-6
Template
strand
RNA primer
for fragment 1
Okazaki
fragment 1
RNA primer
for fragment 2
Okazaki
fragment 2
Overall direction of replication
3
3
3
3
3
3
3
3
3
3
3
3
5
5
5
5
5
5
5
5
5
5
5
5
2
2
2
1
1
1
1
1

Figure 16.17
Overview
Leading
strand
Origin of
replication Lagging
strand
Leading
strand
Lagging
strand Overall directions
of replication
Leading strand
DNA pol III
DNA pol III Lagging strand
DNA pol I DNA ligase
Primer
Primase
Parental
DNA
5
5
5
5
5
3
3
3
3
3
3 2 1
4

Figure 16.17a
Overview
Leading
strand
Origin of
replication Lagging
strand
Leading
strand
Lagging
of replication
Leading strand
DNA pol III
Primer
Primase
Parental
DNA
5
5
3
3
3

Overview
Leading
strand
Origin of
replication Lagging
strand
Leading
strand
Lagging
of replication
Leading strand
Primer
DNA pol III
DNA pol I
Lagging strand
DNA ligase
5
5
5
3
3
3 3
4
2 1
Figure 16.17b

The DNA Replication Complex
• The proteins that participate in DNA replication
form a large complex, a “DNA replication machine”
• The DNA replication machine may be 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

Figure 16.18
Parental DNA
DNA pol III
Leading strand
Connecting
protein
Helicase
Lagging strand
DNA
pol III
Lagging
strand
template
5
5
5
5
5
5
3 3
3
3
3
3

Proofreading and Repairing DNA
• 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 exposure to harmful
chemical or physical agents such as cigarette
smoke and X-rays; it can also undergo
spontaneous changes
• In nucleotide excision repair, a nuclease cuts
out and replaces damaged stretches of DNA

Figure 16.19
Nuclease
DNA
polymerase
DNA
ligase
5
5
5
5
5
5
5
5
3
3
3
3
3
3
3
3

Evolutionary Significance of Altered
DNA Nucleotides
• Error rate after proofreading repair is low but not
zero
• Sequence changes may become permanent and
can be passed on to the next generation
• These changes (mutations) are the source of the
genetic variation upon which natural selection
operates

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 with
uneven ends
• This is not a problem for prokaryotes, most of
which have circular chromosomes

Figure 16.20
Ends of parental
DNA strands
Leading strand
Lagging strand
Last fragment Next-to-last fragment
Lagging strand RNA primer
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
Second round
of replication
Further rounds
of replication
New leading strand
New lagging strand
Shorter and shorter daughter molecules
3
3
3
3
3
5
5
5
5
5

Figure 16.20a
Ends of parental
DNA strands
Leading strand
Lagging strand
Last fragment Next-to-last fragment
Lagging strand RNA primer
Parental strand
Removal of primers and
replacement with DNA
where a 3 end is available
3
3
3
5
5
5

Figure 16.20b
Second round
of replication
Further rounds
of replication
New leading strand
New lagging strand
Shorter and shorter daughter molecules
3
3
3
5
5
5

• Eukaryotic chromosomal DNA molecules have
special nucleotide sequences at their ends called
telomeres
• Telomeres do not prevent the shortening of DNA
molecules, but they do postpone the erosion of
genes near the ends of DNA molecules
• It has been proposed that the shortening of
telomeres is connected to aging

• 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

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

• Chromatin, a complex of DNA and protein,
is found in the nucleus of eukaryotic cells
• Chromosomes fit into the nucleus through
an elaborate, multilevel system of packing

Figure 16.22a
DNA double helix
(2 nm in diameter)
DNA, the double helix
Nucleosome
(10 nm in diameter)
Histones
Histones
Histone
tail
H1
Nucleosomes, or “beads on
a string” (10-nm fiber)

Figure 16.22b
30-nm fiber
30-nm fiber
Loops Scaffold
300-nm fiber
Chromatid
(700 nm)
Replicated
chromosome
(1,400 nm)
Looped domains
(300-nm fiber) Metaphase
chromosome

Figure 16.22c
DNA double helix (2 nm in diameter)

Figure 16.22d
Nucleosome (10 nm in diameter)

Figure 16.22g
Chromatid
(700 nm)

• Chromatin undergoes changes in packing during
the cell cycle
• At interphase, some chromatin is organized into a
10-nm fiber, but much is compacted into a 30-nm
fiber, through folding and looping
• Though interphase chromosomes are not highly
condensed, they still occupy specific restricted
regions in the nucleus

• 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

Figure 16.UN03
DNA pol III synthesizes
leading strand continuously
Parental
DNA DNA pol III starts DNA
synthesis at 3 end of primer,
continues in 5  3 direction
Origin of
replication
Helicase
Primase synthesizes
a short RNA primer
DNA pol I replaces the RNA
primer with DNA nucleotides
3
3
3
5
5
5
5
Lagging strand synthesized
in short Okazaki fragments,
later joined by DNA ligase

B- From Gene to Protein

The Flow of Genetic Information
• The information content of DNA is in the
form of specific sequences of nucleotides
• The DNA inherited by an organism leads to
specific traits by dictating the synthesis of
proteins
• Proteins are the links between genotype
and phenotype
• Gene expression, the process by which
DNA directs protein synthesis, includes two
stages: transcription and translation

Genes specify proteins via transcription
and translation
• How was the fundamental relationship
between genes and proteins discovered?

Evidence from the Study of Metabolic
Defects
• In 1902, British physician Archibald Garrod
first suggested that genes dictate
phenotypes through enzymes that catalyze
specific chemical reactions
• He thought symptoms of an inherited
disease reflect an inability to synthesize a
certain enzyme
• Linking genes to enzymes required
understanding that cells synthesize and

Nutritional Mutants in Neurospora:
Scientific Inquiry
• George Beadle and Edward Tatum exposed
bread mold to X-rays, creating mutants that
were unable to survive on minimal media
• Using crosses, they and their coworkers
identified three classes of arginine-deficient
mutants, each lacking a different enzyme
necessary for synthesizing arginine
• They developed a one gene–one enzyme
hypothesis, which states that each gene
dictates production of a specific enzyme

Figure 17.2
Minimal medium
No growth:
Mutant cells
cannot grow
and divide
Growth:
Wild-type
cells growing
and dividing
EXPERIMENT RESULTS
CONCLUSION
Classes of Neurospora crassa
Wild type Class I mutants Class II mutants Class III mutants
Minimal
medium
(MM)
(control)
MM 
ornithine
MM 
citrulline
Condition
MM 
arginine
(control)
Summary
of results
Can grow with
or without any
supplements
Can grow on
ornithine,
citrulline, or
arginine
Can grow only
on citrulline or
arginine
Require arginine
to grow
Wild type
Class I mutants
(mutation in
gene A)
Class II mutants
(mutation in
gene B)
Class III mutants
(mutation in
gene C)
Gene
(codes for
enzyme)
Gene A
Gene B
Gene C
Precursor Precursor Precursor Precursor
Enzyme A Enzyme A Enzyme A Enzyme A
Enzyme B Enzyme B Enzyme B Enzyme B
Enzyme C Enzyme C Enzyme C Enzyme C
Ornithine Ornithine Ornithine Ornithine
Citrulline Citrulline Citrulline Citrulline
Arginine Arginine Arginine Arginine

Figure 17.2a
Minimal medium
No growth:
Mutant cells
cannot grow
and divide
Growth:
Wild-type
cells growing
and dividing
EXPERIMENT

Figure 17.2b
RESULTS
Classes of Neurospora crassa
Wild type Class I mutants Class II mutants Class III mutants
Minimal
medium
(MM)
(control)
MM 
ornithine
MM 
citrulline
Condition
MM 
arginine
(control)
Summary
of results
Can grow with
or without any
supplements
Can grow on
ornithine,
citrulline, or
arginine
Can grow only
on citrulline or
arginine
Require arginine
to grow
Growth
No
growth

Figure 17.2c
CONCLUSION
Wild type
Class I mutants
(mutation in
gene A)
Class II mutants
(mutation in
gene B)
Class III mutants
(mutation in
gene C)
Gene
(codes for
enzyme)
Gene A
Gene B
Gene C
Precursor Precursor Precursor Precursor
Enzyme A Enzyme A Enzyme A Enzyme A
Enzyme B Enzyme B Enzyme B Enzyme B
Ornithine Ornithine Ornithine Ornithine
Enzyme C Enzyme C Enzyme C
Enzyme C
Citrulline Citrulline Citrulline Citrulline
Arginine Arginine Arginine Arginine

The Products of Gene Expression:
A Developing Story
• Some proteins aren’t enzymes, so
researchers later revised the hypothesis:
one gene–one protein
• Many proteins are composed of several
polypeptides, each of which has its own
gene
• Therefore, Beadle and Tatum’s hypothesis
is now restated as the one gene–one
polypeptide hypothesis
• Note that it is common to refer to gene

Basic Principles of Transcription and
Translation
• RNA is the bridge between genes and the
proteins for which they code
• Transcription is the synthesis of RNA
using information in DNA
• Transcription produces messenger RNA
(mRNA)
• Translation is the synthesis of a
polypeptide, using information in the
mRNA

• In prokaryotes, translation of mRNA can
begin before transcription has finished
• In a eukaryotic cell, the nuclear envelope
separates transcription from translation
• Eukaryotic RNA transcripts are modified
through RNA processing to yield the
finished mRNA

• A primary transcript is the initial RNA
transcript from any gene prior to
processing
• The central dogma is the concept that
cells are governed by a cellular chain of
command: DNA RNA protein

Figure 17.UN01
DNA RNA Protein

Figure 17.3
DNA
mRNA
Ribosome
Polypeptide
TRANSCRIPTION
TRANSLATION
TRANSCRIPTION
TRANSLATION
Polypeptide
Ribosome
DNA
mRNA
Pre-mRNA
RNA PROCESSING
(a) Bacterial cell (b) Eukaryotic cell
Nuclear
envelope

Figure 17.3a-1
TRANSCRIPTION
DNA
mRNA
(a) Bacterial cell

Figure 17.3a-2
TRANSCRIPTION
DNA
mRNA
(a) Bacterial cell
TRANSLATION
Ribosome
Polypeptide

Figure 17.3b-1
Nuclear
envelope
DNA
Pre-mRNA
(b) Eukaryotic cell
TRANSCRIPTION

Figure 17.3b-2
RNA PROCESSING
Nuclear
envelope
DNA
Pre-mRNA
(b) Eukaryotic cell
mRNA
TRANSCRIPTION

Figure 17.3b-3
RNA PROCESSING
Nuclear
envelope
DNA
Pre-mRNA
(b) Eukaryotic cell
mRNA
TRANSCRIPTION
TRANSLATION Ribosome
Polypeptide

The Genetic Code
• How are the instructions for assembling
amino acids into proteins encoded into
DNA?
• There are 20 amino acids, but there are only
four nucleotide bases in DNA
• How many nucleotides correspond to an
amino acid?

Codons: Triplets of Nucleotides
• The flow of information from gene to protein
is based on a triplet code: a series of
nonoverlapping, three-nucleotide words
• The words of a gene are transcribed into
complementary nonoverlapping three-
nucleotide words of mRNA
• These words are then translated into a
chain of amino acids, forming a polypeptide

Figure 17.4
DNA
template
strand
TRANSCRIPTION
mRNA
TRANSLATION
Protein
Amino acid
Codon
Trp Phe Gly
5
5
Ser
U U U U U
3
3
5
3
G
G
G G C C
T
C
A
A
A
A
A
A
A
T T T T
T
G
G G G
C C C G G
DNA
molecule
Gene 1
Gene 2
Gene 3
C C

• During transcription, one of the two DNA
strands, called the template strand,
provides a template for ordering the
sequence of complementary nucleotides in
an RNA transcript
• The template strand is always the same
strand for a given gene
• During translation, the mRNA base triplets,
called codons, are read in the 5 to 3
direction

• Codons along an mRNA molecule are read
by translation machinery in the 5 to 3
direction
• Each codon specifies the amino acid (one
of 20) to be placed at the corresponding
position along a polypeptide

Cracking the Code
• All 64 codons were deciphered by the mid-
1960s
• Of the 64 triplets, 61 code for amino acids;
3 triplets are “stop” signals to end
translation
• The genetic code is redundant (more than
one codon may specify a particular amino
acid) but not ambiguous; no codon
specifies more than one amino acid
• Codons must be read in the correct
reading frame (correct groupings) in order

Figure 17.5
Second mRNA base
First
mRNA
base
(5
end
of
codon)
Third
mRNA
base
(3
end
of
codon)
UUU
UUC
UUA
CUU
CUC
CUA
CUG
Phe
Leu
Leu
Ile
UCU
UCC
UCA
UCG
Ser
CCU
CCC
CCA
CCG
UAU
UAC
Tyr
Pro
Thr
UAA Stop
UAG Stop
UGA Stop
UGU
UGC
Cys
UGG Trp
G
C
U
U
C
A
U
U
C
C
C
A
U
A
A
A
G
G
His
Gln
Asn
Lys
Asp
CAU CGU
CAC
CAA
CAG
CGC
CGA
CGG
G
AUU
AUC
AUA
ACU
ACC
ACA
AAU
AAC
AAA
AGU
AGC
AGA
Arg
Ser
Arg
Gly
ACG
AUG AAG AGG
GUU
GUC
GUA
GUG
GCU
GCC
GCA
GCG
GAU
GAC
GAA
GAG
Val Ala
GGU
GGC
GGA
GGG
Glu
Gly
G
U
C
A
Met or
start
UUG
G

Evolution of the Genetic Code
• The genetic code is nearly universal, shared
by the simplest bacteria to the most
complex animals
• Genes can be transcribed and translated
after being transplanted from one species to
another

Figure 17.6
(a) Tobacco plant expressing
a firefly gene gene
(b) Pig expressing a jellyfish

Figure 17.6a
(a) Tobacco plant expressing
a firefly gene

Figure 17.6b
(b) Pig expressing a jellyfish
gene

Transcription is the DNA-directed
synthesis of RNA: a closer look
• Transcription is the first stage of gene
expression

Molecular Components of Transcription
• RNA synthesis is catalyzed by RNA
polymerase, which pries the DNA strands
apart and hooks together the RNA
nucleotides
• The RNA is complementary to the DNA
template strand
• RNA synthesis follows the same base-
pairing rules as DNA, except that uracil
substitutes for thymine

• The DNA sequence where RNA polymerase
attaches is called the promoter; in bacteria,
the sequence signaling the end of
transcription is called the terminator
• The stretch of DNA that is transcribed is
called a transcription unit

Figure 17.7-1 Promoter
RNA polymerase
Start point
DNA
5
3
Transcription unit
3
5

RNA polymerase
Start point
DNA
5
3
Transcription unit
3
5
Initiation
5
3
3
5
Nontemplate strand of DNA
Template strand of DNA
RNA
transcript
Unwound
DNA
1

RNA polymerase
Start point
DNA
5
3
Transcription unit
3
5
Elongation
5
3
3
5
RNA
transcript
Unwound
DNA
2
3
5
3
5
3
Rewound
DNA
RNA
transcript
5
Initiation
1

RNA polymerase
Start point
DNA
5
3
Transcription unit
3
5
Elongation
5
3
3
5
RNA
transcript
Unwound
DNA
2
3
5
3
5
3
Rewound
DNA
RNA
transcript
5
Termination
3
3
5
5
Completed RNA transcript
Direction of transcription (“downstream”)
5
3
3
Initiation
1

Synthesis of an RNA Transcript
• The three stages of transcription
– Initiation
– Elongation
– Termination

RNA Polymerase Binding and Initiation
of Transcription
• Promoters signal the transcriptional start
point and usually extend several dozen
nucleotide pairs upstream of the start point
• Transcription factors mediate the binding
of RNA polymerase and the initiation of
transcription
• The completed assembly of transcription
factors and RNA polymerase II bound to a
promoter is called a transcription initiation
complex

Figure 17.8
Transcription initiation
complex forms
3
DNA
Promoter
Nontemplate strand
5
3
5
3
5
3
Transcription
factors
RNA polymerase II
Transcription factors
5
3
5
3
5
3
RNA transcript
Transcription initiation complex
5
3
TATA box
T
T T T T T
A A A AA
A A
T
Several transcription
factors bind to DNA
2
A eukaryotic promoter
1
Start point Template strand

Elongation of the RNA Strand
• As RNA polymerase moves along the
DNA, it untwists the double helix, 10 to 20
bases at a time
• Transcription progresses at a rate of 40
nucleotides per second in eukaryotes
• A gene can be transcribed simultaneously
by several RNA polymerases
• Nucleotides are added to the 3 end of the
growing RNA molecule

Nontemplate
strand of DNA
RNA nucleotides
RNA
polymerase
Template
strand of DNA
3
3
5
5
5
3
Newly made
RNA
Direction of transcription
A A A
A
T
T
T
T G
C
C C
G
C C
C A A
U
end
Figure 17.9

Termination of Transcription
• The mechanisms of termination are different
in bacteria and eukaryotes
• In bacteria, the polymerase stops
transcription at the end of the terminator and
the mRNA can be translated without further
modification
• In eukaryotes, RNA polymerase II
transcribes the polyadenylation signal
sequence; the RNA transcript is released
10–35 nucleotides past this polyadenylation

Eukaryotic cells modify RNA after
transcription
• Enzymes in the eukaryotic nucleus modify
pre-mRNA (RNA processing) before the
genetic messages are dispatched to the
cytoplasm
• During RNA processing, both ends of the
primary transcript are usually altered
• Also, usually some interior parts of the
molecule are cut out, and the other parts
spliced together

Alteration of mRNA Ends
• Each end of a pre-mRNA molecule is
modified in a particular way
– The 5 end receives a modified nucleotide 5
cap
– The 3 end gets a poly-A tail
• These modifications share several
functions
– They seem to facilitate the export of mRNA
to the cytoplasm
– They protect mRNA from hydrolytic
enzymes
– They help ribosomes attach to the 5 end

Figure 17.10
Protein-coding
segment
Polyadenylation
signal
5 3
3
5 5
Cap UTR
Start
codon
G P P P
Stop
codon
UTR
AAUAAA
Poly-A tail
AAA AAA
…

Split Genes and RNA Splicing
• Most eukaryotic genes and their RNA
transcripts have long noncoding stretches of
nucleotides that lie between coding regions
• These noncoding regions are called
intervening sequences, or introns
• The other regions are called exons because
they are eventually expressed, usually
translated into amino acid sequences
• RNA splicing removes introns and joins
exons, creating an mRNA molecule with a
continuous coding sequence

Figure 17.11
5 Exon Intron Exon
5 Cap
Pre-mRNA
Codon
numbers
130 31104
mRNA 5Cap
5
Intron Exon
3 UTR
Introns cut out and
exons spliced together
3
105
146
Poly-A tail
Coding
segment
Poly-A tail
UTR
1146

• In some cases, RNA splicing is carried out
by spliceosomes
• Spliceosomes consist of a variety of
proteins and several small nuclear
ribonucleoproteins (snRNPs) that
recognize the splice sites

Figure 17.12-1
RNA transcript (pre-mRNA)
5
Exon 1
Protein
snRNA
snRNPs
Intron Exon 2
Other
proteins

Figure 17.12-2
5
Exon 1
Protein
snRNA
snRNPs
Intron Exon 2
Other
proteins
Spliceosome
5

Figure 17.12-3
5
Exon 1
Protein
snRNA
snRNPs
Intron Exon 2
Other
proteins
Spliceosome
5
Spliceosome
components
Cut-out
intron
mRNA
5
Exon 1 Exon 2

Ribozymes
• Ribozymes are catalytic RNA molecules
that function as enzymes and can splice
RNA
• The discovery of ribozymes rendered
obsolete the belief that all biological
catalysts were proteins

• Three properties of RNA enable it to
function as an enzyme
– It can form a three-dimensional structure
because of its ability to base-pair with itself
– Some bases in RNA contain functional groups
that may participate in catalysis
– RNA may hydrogen-bond with other nucleic
acid molecules

The Functional and Evolutionary
Importance of Introns
• Some introns contain sequences that may
regulate gene expression
• Some genes can encode more than one
kind of polypeptide, depending on which
segments are treated as exons during
splicing
• This is called alternative RNA splicing
• Consequently, the number of different
proteins an organism can produce is much

• Proteins often have a modular architecture
consisting of discrete regions called
domains
• In many cases, different exons code for the
different domains in a protein
• Exon shuffling may result in the evolution of
new proteins

Gene
DNA
Exon 1 Exon 2 Exon 3
Intron Intron
Transcription
RNA processing
Translation
Domain 3
Domain 2
Domain 1
Polypeptide
Figure 17.13

Translation is the RNA-directed
synthesis of a polypeptide: a closer look
• Genetic information flows from mRNA to
protein through the process of translation

Molecular Components of Translation
• A cell translates an mRNA message into
protein with the help of transfer RNA
(tRNA)
• tRNAs transfer amino acids to the growing
polypeptide in a ribosome
• Translation is a complex process in terms of
its biochemistry and mechanics

Figure 17.14
Polypeptide
Ribosome
tRNA with
amino acid
attached
Amino
acids
tRNA
Anticodon
Codons
U U U U
G G G G C
C
G
C
G
5 3
mRNA

The Structure and Function of Transfer
RNA
Molecules of tRNA are not identical
– Each carries a specific amino acid on one end
– Each has an anticodon on the other end; the
anticodon base-pairs with a complementary
codon on mRNA

• A tRNA molecule consists of a single RNA
strand that is only about 80 nucleotides
long
• Flattened into one plane to reveal its base
pairing, a tRNA molecule looks like a
cloverleaf

Figure 17.15
Amino acid
attachment
site
3
5
Hydrogen
bonds
Anticodon
(a) Two-dimensional structure (b) Three-dimensional structure
(c) Symbol used
in this book
Anticodon Anticodon
3 5
Hydrogen
bonds
Amino acid
attachment
site
5
3
A A G

Figure 17.15a
Amino acid
attachment
site
3
5
Hydrogen
bonds
Anticodon
(a) Two-dimensional structure

(b) Three-dimensional structure
(c) Symbol used
Anticodon Anticodon
3 5
Hydrogen
bonds
Amino acid
attachment
site
5
3
in this book
A A G
Figure 17.15b

• Because of hydrogen bonds, tRNA
actually twists and folds into a three-
dimensional molecule
• tRNA is roughly L-shaped

• Accurate translation requires two steps
– First: a correct match between a tRNA and an
amino acid, done by the enzyme aminoacyl-
tRNA synthetase
– Second: a correct match between the tRNA
anticodon and an mRNA codon
• Flexible pairing at the third base of a codon
is called wobble and allows some tRNAs to
bind to more than one codon

Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P Adenosine
ATP
Figure 17.16-1

Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P Adenosine
ATP
P
P
P
P
P
i
i
i
Adenosine
Figure 17.16-2

Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P Adenosine
ATP
P
P
P
P
P
i
i
i
Adenosine
tRNA
Adenosine
P
tRNA
AMP
Computer model
Amino
acid
Aminoacyl-tRNA
synthetase
Figure 17.16-3

Aminoacyl-tRNA
synthetase (enzyme)
Amino acid
P P P Adenosine
ATP
P
P
P
P
P
i
i
i
Adenosine
tRNA
Adenosine
P
tRNA
AMP
Computer model
Amino
acid
Aminoacyl-tRNA
synthetase
Aminoacyl tRNA
(“charged tRNA”)
Figure 17.16-4

Ribosomes
• Ribosomes facilitate specific coupling of
tRNA anticodons with mRNA codons in
protein synthesis
• The two ribosomal subunits (large and small)
are made of proteins and ribosomal RNA
(rRNA)
• Bacterial and eukaryotic ribosomes are
somewhat similar but have significant
differences: some antibiotic drugs
specifically target bacterial ribosomes

tRNA
molecules
Growing
polypeptide Exit tunnel
E P
A
Large
subunit
Small
subunit
mRNA
5
3
(a) Computer model of functioning ribosome
Exit tunnel Amino end
A site (Aminoacyl-
tRNA binding site)
Small
subunit
Large
subunit
E P A
mRNA
E
P site (Peptidyl-tRNA
binding site)
mRNA
binding site
(b) Schematic model showing binding sites
E site
(Exit site)
(c) Schematic model with mRNA and tRNA
5 Codons
3
tRNA
Growing polypeptide
Next amino
acid to be
added to
polypeptide
chain
Figure 17.17

Figure 17.17a
tRNA
molecules
Growing
polypeptide Exit tunnel
E P A
Large
subunit
Small
subunit
mRNA
5
3
(a) Computer model of functioning ribosome

Figure 17.17b
Exit tunnel
A site (Aminoacyl-
tRNA binding site)
Small
subunit
Large
subunit
P A
P site (Peptidyl-tRNA
binding site)
mRNA
binding site
(b) Schematic model showing binding sites
E site
(Exit site)
E

Figure 17.17c
Amino end
mRNA
E
(c) Schematic model with mRNA and tRNA
5 Codons
3
tRNA
Growing polypeptide
Next amino
acid to be
added to
polypeptide
chain

• A ribosome has three binding sites for tRNA
– The P site holds the tRNA that carries the
growing polypeptide chain
– The A site holds the tRNA that carries the
next amino acid to be added to the chain
– The E site is the exit site, where discharged
tRNAs leave the ribosome

Building a Polypeptide
• The three stages of translation
– Initiation
– Elongation
– Termination
• All three stages require protein “factors” that
aid in the translation process

Ribosome Association and Initiation of
Translation
• The initiation stage of translation brings
together mRNA, a tRNA with the first amino
acid, and the two ribosomal subunits
• First, a small ribosomal subunit binds with
mRNA and a special initiator tRNA
• Then the small subunit moves along the
mRNA until it reaches the start codon (AUG)
• Proteins called initiation factors bring in the
large subunit that completes the translation
initiation complex

Figure 17.18
Initiator
tRNA
mRNA
5
5
3
Start codon
Small
ribosomal
subunit
mRNA binding site
3
Translation initiation complex
5 3
3 U
U
A
A G
C
P
P site
i

GTP GDP
Large
ribosomal
subunit
E A
5

Elongation of the Polypeptide Chain
• During the elongation stage, amino acids
are added one by one to the preceding
amino acid at the C-terminus of the
growing chain
• Each addition involves proteins called
elongation factors and occurs in three
steps: codon recognition, peptide bond
formation, and translocation
• Translation proceeds along the mRNA in a
5′ to 3′ direction

Amino end of
polypeptide
mRNA
5
E
P
site
A
site
3
Figure 17.19-1

Amino end of
polypeptide
mRNA
5
E
P
site
A
site
3
E
GTP
GDP  P i
P A
Figure 17.19-2

Amino end of
polypeptide
mRNA
5
E
P
site
A
site
3
E
GTP
GDP  P i
P A
E
P A
Figure 17.19-3

Amino end of
polypeptide
mRNA
5
E
A
site
3
E
GTP
GDP  P i
P A
E
P A
GTP
GDP  P i
P A
E
Ribosome ready for
next aminoacyl tRNA
P
site
Figure 17.19-4

Termination of Translation
• Termination occurs when a stop codon in
the mRNA reaches the A site of the
ribosome
• The A site accepts a protein called a
release factor
• The release factor causes the addition of a
water molecule instead of an amino acid
• This reaction releases the polypeptide,
and the translation assembly then comes
apart

Figure 17.20-1
Release
factor
Stop codon
(UAG, UAA, or UGA)
3
5

Figure 17.20-2
Release
factor
Stop codon
(UAG, UAA, or UGA)
3
5
3
5
Free
polypeptide
2 GTP
2 GDP  2 i
P

Figure 17.20-3
Release
factor
Stop codon
(UAG, UAA, or UGA)
3
5
3
5
Free
polypeptide
2 GTP
5
3
2 GDP  2 i
P

Polyribosomes
• A number of ribosomes can translate a
single mRNA simultaneously, forming a
polyribosome (or polysome)
• Polyribosomes enable a cell to make many
copies of a polypeptide very quickly

Figure 17.21
Completed
polypeptide
Incoming
ribosomal
subunits
Start of
mRNA
(5 end)
End of
mRNA
(3 end)
(a)
Ribosomes
mRNA
(b)
0.1 m
Growing
polypeptides

Figure 17.21a
Ribosomes
mRNA
0.1 m

Completing and Targeting the Functional
Protein
• Often translation is not sufficient to make a
functional protein
• Polypeptide chains are modified after
translation or targeted to specific sites in the
cell

Protein Folding and Post-Translational
Modifications
• During and after synthesis, a polypeptide
chain spontaneously coils and folds into its
three-dimensional shape
• Proteins may also require post-
translational modifications before doing
their job
• Some polypeptides are activated by
enzymes that cleave them
• Other polypeptides come together to form

Targeting Polypeptides to Specific Locations
• Two populations of ribosomes are evident
in cells: free ribsomes (in the cytosol) and
bound ribosomes (attached to the ER)
• Free ribosomes mostly synthesize proteins
that function in the cytosol
• Bound ribosomes make proteins of the
endomembrane system and proteins that
are secreted from the cell
• Ribosomes are identical and can switch
from free to bound

• Polypeptide synthesis always begins in
the cytosol
• Synthesis finishes in the cytosol unless
the polypeptide signals the ribosome to
attach to the ER
• Polypeptides destined for the ER or for
secretion are marked by a signal peptide

• A signal-recognition particle (SRP)
binds to the signal peptide
• The SRP brings the signal peptide and its
ribosome to the ER

Figure 17.22
Ribosome
mRNA
Signal
peptide
SRP
1
SRP
receptor
protein
Translocation
complex
ER
LUMEN
2
3
4
5
6
Signal
peptide
removed
CYTOSOL
Protein
ER
membrane

Concept 17.5: Mutations of one or a few
nucleotides can affect protein structure and
function
• Mutations are changes in the genetic
material of a cell or virus
• Point mutations are chemical changes in
just one base pair of a gene
• The change of a single nucleotide in a
DNA template strand can lead to the
production of an abnormal protein

Figure 17.23
Wild-type hemoglobin
Wild-type hemoglobin DNA
3
3
3
5
5 3
3
5
5
5
5
3
mRNA
A A
G
C T T
A A
G
mRNA
Normal hemoglobin
Glu
Sickle-cell hemoglobin
Val
A
A
A
U
G
G
T
T
Sickle-cell hemoglobin
Mutant hemoglobin DNA
C

Types of Small-Scale Mutations
• Point mutations within a gene can be
divided into two general categories
– Nucleotide-pair substitutions
– One or more nucleotide-pair insertions or
deletions

Substitutions
• A nucleotide-pair substitution replaces
one nucleotide and its partner with
another pair of nucleotides
• Silent mutations have no effect on the
amino acid produced by a codon because
of redundancy in the genetic code
• Missense mutations still code for an
amino acid, but not the correct amino acid
• Nonsense mutations change an amino
acid codon into a stop codon, nearly
always leading to a nonfunctional protein

Wild type
DNA template strand
mRNA5
5
3
Protein
Amino end
A instead of G
(a) Nucleotide-pair substitution
3
3
5
Met Lys Phe Gly Stop
Carboxyl end
T T T T T
T
T
T
T
T
A A A A A
A
A
A
A
C
C
C
C
A
A A A A A
G G G G
G
C C
G G
G
U U U U U
G
(b) Nucleotide-pair insertion or deletion
Extra A
3
5
5
3
Extra U
5 3
T T T T
T T T T
A
A A A
A
A
T G G G G
G
A
A
A
A
C
C
C
C
C A
T
3
5
5 3
5
T T T T T
A
A
A
A
C
C
A A
C C
T
T
T
T
T
A A A A A
T
G G G G
U instead of C
Stop
U
A A A A A
G G
G
U U U U U
G
Met
Lys Phe Gly
Silent (no effect on amino acid sequence)
T instead of C
T T T T T
A
A
A
A
C
C
A G
T C
T A T T T
A
A
A
A
C
C
A G
C C
A instead of G
C
A A A A A
G A
G
U U U U U
G U
A A A A
G G
G
U U U G A
C
A
A U U A A
U U
G
U G G C U
A
G
A U A U A
A U
G
U G U U C
G
Met Lys Phe Ser
Stop
Stop Met Lys
missing
missing
Frameshift causing immediate nonsense
(1 nucleotide-pair insertion)
Frameshift causing extensive missense
(1 nucleotide-pair deletion)
missing
T T T T T
T
C
A
A
C
C
A A
C G
A
G
T
T
T
A A A A A
T
G G G C
Leu Ala
Missense
A instead of T
T
T
T
T
T
A A A A A
C
G G A G
A
C
A U A A A
G G
G
U U U U U
G
T
T
T
T
T
A T A A A
C
G G G G
Met
Nonsense
Stop
U instead of A
3
5
3
5
5
3
3
5
5
3
3
5 3
Met Phe Gly
No frameshift, but one amino acid missing
(3 nucleotide-pair deletion)
missing
3
5
5
3
5 3
U
T C
A A
A C
A T
T
A
C G
T
A G T T T G G A A
T
C
T T C
A A G
Met
3
T
A
Stop
3
5
5
3
5 3
Figure 17.24

Figure 17.24a
Wild type
DNA template strand
mRNA5
5
Protein
Amino end
Stop
Carboxyl end
3
3
3
5
Met Lys Phe Gly
A instead of G
(a) Nucleotide-pair substitution: silent
Stop
Met Lys Phe Gly
U instead of C
A
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
C
C
G G G G
G
G
A
A A A A
G G
G
U U U U U
5
3
3
5
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
G G G G
A
A
A G A A A A
G G
G
U U U U U
T
U 3
5

Figure 17.24b
Wild type
DNA template strand
mRNA5
5
Protein
Amino end
Stop
Carboxyl end
3
3
3
5
Met Lys Phe Gly
T instead of C
(a) Nucleotide-pair substitution: missense
Stop
Met Lys Phe Ser
A instead of G
A
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
C
C
G G G G
G
G
A
A A A A
G G
G
U U U U U
5
3
3
5
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C T C
G
G
G
A
A G A A A A
A G
G
U U U U U 3
5
A C
C
G

Figure 17.24c
Wild type
DNA template strand
mRNA5
5
Protein
Amino end
Stop
Carboxyl end
3
3
3
5
Met Lys Phe Gly
A instead of T
(a) Nucleotide-pair substitution: nonsense
Met
A
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
C
C
G G G G
G
G
A
A A A A
G G
G
U U U U U
5
3
3
5
A
A
A A A A
A A
T
T A T T T
T T T
T
C C C
G
G
G
A
A G U A A A
G
G
U U U U U 3
5
C
C
G
T instead of C
C
G
T
U instead of A
G
Stop

Insertions and Deletions
• Insertions and deletions are additions or
losses of nucleotide pairs in a gene
• These mutations have a disastrous effect on
the resulting protein more often than
substitutions do
• Insertion or deletion of nucleotides may alter
the reading frame, producing a frameshift
mutation

Figure 17.24d
Wild type
DNA template strand
mRNA5
5
Protein
Amino end
Stop
Carboxyl end
3
3
3
5
Met Lys Phe Gly
A
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
C
C
G G G G
G
G
A
A A A A
G G
G
U U U U U
(b) Nucleotide-pair insertion or deletion: frameshift causing
immediate nonsense
Extra A
Extra U
5
3
5
3
3
5
Met
1 nucleotide-pair insertion
Stop
A C A A G
T T A T
C T A C G
T A T A
T G T C
T G
G A T G
A
A G U A U A
U G
A
U G U U C
A T
A
A
G

Figure 17.24e
DNA template strand
mRNA5
5
Protein
Amino end
Stop
Carboxyl end
3
3
3
5
Met Lys Phe Gly
A
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
C
C
G G G G
G
G
A
A A A A
G G
G
U U U U U
(b) Nucleotide-pair insertion or deletion: frameshift causing
extensive missense
Wild type
missing
missing
A
U
A A A
T T T
C C A T T
C C G
A A
T T T
G G
A A A
T
C
G G
A G A A G
U U U C A A
G G U 3
5
3
3
5
Met Lys Leu Ala
1 nucleotide-pair deletion
5

Figure 17.24f
DNA template strand
mRNA5
5
Protein
Amino end
Stop
Carboxyl end
3
3
3
5
Met Lys Phe Gly
A
A
A A
A A A A
A A
T
T T T T T
T T T
T
C C C C
C
C
G G G G
G
G
A
A A A A
G G
G
U U U U U
(b) Nucleotide-pair insertion or deletion: no frameshift, but one
amino acid missing
Wild type
A
T C A A A A T T
C C G
T T C missing
missing
Stop
5
3
3
5
3
5
Met Phe Gly
3 nucleotide-pair deletion
A G
U C A A
G G
U U U U
T G
A A A
T T T
T C
G G
A A G

Mutagens
• Spontaneous mutations can occur during
DNA replication, recombination, or repair
• Mutagens are physical or chemical agents
that can cause mutations

Concept 17.6: While gene expression differs
among the domains of life, the concept of a
gene is universal
• Archaea are prokaryotes, but share many
features of gene expression with
eukaryotes

Comparing Gene Expression in
Bacteria, Archaea, and Eukarya
• Bacteria and eukarya differ in their RNA
polymerases, termination of transcription,
and ribosomes; archaea tend to resemble
eukarya in these respects
• Bacteria can simultaneously transcribe
and translate the same gene
• In eukarya, transcription and translation
are separated by the nuclear envelope
• In archaea, transcription and translation
are likely coupled

Figure 17.25
RNA polymerase
DNA
mRNA
Polyribosome
RNA
polymerase DNA
Polyribosome
Polypeptide
(amino end)
mRNA (5 end)
Ribosome
0.25 m
Direction of
transcription

Figure 17.25a
RNA polymerase
DNA
mRNA
Polyribosome
0.25 m

What Is a Gene? Revisiting the Question
• The idea of the gene has evolved through
the history of genetics
• We have considered a gene as
– A discrete unit of inheritance
– A region of specific nucleotide sequence in a
chromosome
– A DNA sequence that codes for a specific
polypeptide chain

Figure 17.26
TRANSCRIPTION
DNA
RNA
polymerase
Exon
RNA
transcript
RNA
PROCESSING
NUCLEUS
Intron
RNA transcript
(pre-mRNA)
Aminoacyl-
tRNA synthetase
AMINO ACID
ACTIVATION
Amino
acid
tRNA
3
Growing
polypeptide
mRNA
Aminoacyl
(charged)
tRNA
Anticodon
Ribosomal
subunits
A
A
E
TRANSLATION
CYTOPLASM
P
E
Codon
Ribosome
5
3

• In summary, a gene can be defined as a
region of DNA that can be expressed to
produce a final functional product, either a
polypeptide or an RNA molecule

C- Regulation of Gene Expression

Overview: Conducting the Genetic
Orchestra
• Prokaryotes and eukaryotes alter gene
expression in response to their changing
environment
• In multicellular eukaryotes, gene expression
regulates development and is responsible for
differences in cell types
• RNA molecules play many roles in regulating
gene expression in eukaryotes

Bacteria often respond to environmental
change by regulating transcription
• Natural selection has favored bacteria that
produce only the products needed by that cell
• A cell can regulate the production of enzymes by
feedback inhibition or by gene regulation
• Gene expression in bacteria is controlled by the
operon model

Precursor
Feedback
inhibition
Enzyme 1
Enzyme 2
Enzyme 3
Tryptophan
(a) (b)
Regulation of enzyme
activity
Regulation of enzyme
production
Regulation
of gene
expression


trpE gene
trpD gene
trpC gene
trpB gene
trpA gene
Figure 18.2

Operons: The Basic Concept
• A cluster of functionally related genes can be
under coordinated control by a single “on-off
switch”
• The regulatory “switch” is a segment of DNA
called an operator usually positioned within the
promoter
• An operon is the entire stretch of DNA that
includes the operator, the promoter, and the genes
that they control

• The operon can be switched off by a protein
repressor
• The repressor prevents gene transcription by
binding to the operator and blocking RNA
polymerase
• The repressor is the product of a separate
regulatory gene

• The repressor can be in an active or inactive form,
depending on the presence of other molecules
• A corepressor is a molecule that cooperates with
a repressor protein to switch an operon off
• For example, E. coli can synthesize the amino
acid tryptophan

• By default the trp operon is on and the genes for
tryptophan synthesis are transcribed
• When tryptophan is present, it binds to the trp
repressor protein, which turns the operon off
• The repressor is active only in the presence of its
corepressor tryptophan; thus the trp operon is
turned off (repressed) if tryptophan levels are high

Promoter
DNA
Regulatory
gene
mRNA
trpR
5
3
Protein Inactive
repressor
RNA
polymerase
Promoter
trp operon
Genes of operon
Operator
mRNA 5
Start codon Stop codon
trpE trpD trpC trpB trpA
E D C B A
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on
(b) Tryptophan present, repressor active, operon off
DNA
mRNA
Protein
Tryptophan
(corepressor)
Active
repressor
No RNA
made
Figure 18.3

Figure 18.3a
Promoter
DNA
Regulatory
gene
mRNA
trpR
5
3
Protein Inactive
repressor
RNA
polymerase
Promoter
trp operon
Genes of operon
Operator
mRNA 5
Start codon Stop codon
trpE trpD trpC trpB trpA
E D C B A
Polypeptide subunits that make up
enzymes for tryptophan synthesis
(a) Tryptophan absent, repressor inactive, operon on

Figure 18.3b-1
DNA
mRNA
Protein
Tryptophan
(corepressor)
Active
repressor

Figure 18.3b-2
DNA
mRNA
Protein
Tryptophan
(corepressor)
Active
repressor
No RNA
made

Repressible and Inducible Operons:
Two Types of Negative Gene Regulation
• A repressible operon is one that is usually on;
binding of a repressor to the operator shuts off
transcription
• The trp operon is a repressible operon
• An inducible operon is one that is usually off; a
molecule called an inducer inactivates the
repressor and turns on transcription

• The lac operon is an inducible operon and
contains genes that code for enzymes used in the
hydrolysis and metabolism of lactose
• By itself, the lac repressor is active and switches
the lac operon off
• A molecule called an inducer inactivates the
repressor to turn the lac operon on

(a) Lactose absent, repressor active, operon off
(b) Lactose present, repressor inactive, operon on
Regulatory
gene
Promoter
Operator
DNA lacZ
lacI
lacI
DNA
mRNA
5
3
No
RNA
made
RNA
polymerase
Active
repressor
Protein
lac operon
lacZ lacY lacA
DNA
mRNA
5
3
Protein
mRNA 5
Inactive
repressor
RNA polymerase
Allolactose
(inducer)
-Galactosidase Permease Transacetylase
Figure 18.4

Figure 18.4a
(a) Lactose absent, repressor active, operon off
Regulatory
gene
Promoter
Operator
DNA lacZ
lacI
DNA
mRNA
5
3
No
RNA
made
RNA
polymerase
Active
repressor
Protein

Figure 18.4b
(b) Lactose present, repressor inactive, operon on
lacI
lac operon
lacZ lacY lacA
DNA
mRNA
5
3
Protein
mRNA 5
Inactive
repressor
RNA polymerase
Allolactose
(inducer)
-Galactosidase Permease Transacetylase

• Inducible enzymes usually function in catabolic
pathways; their synthesis is induced by a chemical
signal
• Repressible enzymes usually function in anabolic
pathways; their synthesis is repressed by high
levels of the end product
• Regulation of the trp and lac operons involves
negative control of genes because operons are
switched off by the active form of the repressor

Positive Gene Regulation
• Some operons are also subject to positive control
through a stimulatory protein, such as catabolite
activator protein (CAP), an activator of
transcription
• When glucose (a preferred food source of E. coli)
is scarce, CAP is activated by binding with cyclic
AMP (cAMP)
• Activated CAP attaches to the promoter of the lac
operon and increases the affinity of RNA
polymerase, thus accelerating transcription

• When glucose levels increase, CAP detaches from
the lac operon, and transcription returns to a
normal rate
• CAP helps regulate other operons that encode
enzymes used in catabolic pathways

Figure 18.5
Promoter
DNA
CAP-binding site
lacZ
lacI
RNA
polymerase
binds and
transcribes
Operator
cAMP
Active
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized
Promoter
DNA
CAP-binding site
lacZ
lacI
Operator
RNA
polymerase less
likely to bind
Inactive lac
repressor
Inactive
CAP
(b) Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized

Figure 18.5a
Promoter
DNA
CAP-binding site
lacZ
lacI
RNA
polymerase
binds and
transcribes
Operator
cAMP
Active
CAP
Inactive
CAP
Allolactose
Inactive lac
repressor
(a) Lactose present, glucose scarce (cAMP level high):
abundant lac mRNA synthesized

Figure 18.5b
Promoter
DNA
CAP-binding site
lacZ
lacI
Operator
RNA
polymerase less
likely to bind
Inactive lac
repressor
Inactive
CAP
(b) Lactose present, glucose present (cAMP level low):
little lac mRNA synthesized

Eukaryotic gene expression is regulated
at many stages
• All organisms must regulate which genes are
expressed at any given time
• In multicellular organisms regulation of gene
expression is essential for cell specialization

Differential Gene Expression
• Almost all the cells in an organism are genetically
identical
• Differences between cell types result from
differential gene expression, the expression of
different genes by cells with the same genome
• Abnormalities in gene expression can lead to
diseases including cancer
• Gene expression is regulated at many stages

Figure 18.6 Signal
NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene
Gene available
for transcription
RNA Exon
Primary transcript
Transcription
Intron
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM
mRNA in cytoplasm
Translation
Degradation
of mRNA
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Active protein
Degradation
of protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)

Figure 18.6a
Signal
NUCLEUS
Chromatin
Chromatin modification:
DNA unpacking involving
histone acetylation and
DNA demethylation
DNA
Gene
Gene available
for transcription
RNA Exon
Primary transcript
Transcription
Intron
RNA processing
Cap
Tail
mRNA in nucleus
Transport to cytoplasm
CYTOPLASM

Figure 18.6b
CYTOPLASM
mRNA in cytoplasm
Translation
Degradation
of mRNA
Polypeptide
Protein processing, such
as cleavage and
chemical modification
Active protein
Degradation
of protein
Transport to cellular
destination
Cellular function (such
as enzymatic activity,
structural support)

Regulation of Chromatin Structure
• Genes within highly packed heterochromatin are
usually not expressed
• Chemical modifications to histones and DNA of
chromatin influence both chromatin structure and
gene expression

Histone Modifications
• In histone acetylation, acetyl groups are
attached to positively charged lysines in histone
tails
• This loosens chromatin structure, thereby
promoting the initiation of transcription
• The addition of methyl groups (methylation) can
condense chromatin; the addition of phosphate
groups (phosphorylation) next to a methylated
amino acid can loosen chromatin

Figure 18.7
Amino acids
available
for chemical
modification
Histone
tails
DNA
double
helix
Nucleosome
(end view)
(a) Histone tails protrude outward from a nucleosome
Unacetylated histones Acetylated histones
(b) Acetylation of histone tails promotes loose chromatin
structure that permits transcription

• The histone code hypothesis proposes that
specific combinations of modifications, as well as
the order in which they occur, help determine
chromatin configuration and influence transcription

DNA Methylation
• DNA methylation, the addition of methyl groups
to certain bases in DNA, is associated with
reduced transcription in some species
• DNA methylation can cause long-term inactivation
of genes in cellular differentiation
• In genomic imprinting, methylation regulates
expression of either the maternal or paternal
alleles of certain genes at the start of development

Epigenetic Inheritance
• Although the chromatin modifications just
discussed do not alter DNA sequence, they may
be passed to future generations of cells
• The inheritance of traits transmitted by
mechanisms not directly involving the nucleotide
sequence is called epigenetic inheritance

Regulation of Transcription Initiation
• Chromatin-modifying enzymes provide initial
control of gene expression by making a region of
DNA either more or less able to bind the
transcription machinery

Organization of a Typical Eukaryotic Gene
• Associated with most eukaryotic genes are
multiple control elements, segments of
noncoding DNA that serve as binding sites for
transcription factors that help regulate
transcription
• Control elements and the transcription factors they
bind are critical to the precise regulation of gene
expression in different cell types

Figure 18.8-1
Enhancer
(distal control
elements)
DNA
Upstream
Promoter
Proximal
control
elements
Transcription
start site
Exon Intron Exon Exon
Intron
Poly-A
signal
sequence
Transcription
termination
region
Downstream

Figure 18.8-2
Enhancer
(distal control
elements)
DNA
Upstream
Promoter
Proximal
control
elements
Transcription
start site
Intron
Poly-A
signal
sequence
Transcription
termination
region
Downstream
Poly-A
signal
Intron
Transcription
Cleaved
3 end of
primary
transcript
5
Primary RNA
transcript
(pre-mRNA)

Figure 18.8-3
Enhancer
(distal control
elements)
DNA
Upstream
Promoter
Proximal
control
elements
Transcription
start site
Intron
Poly-A
signal
sequence
Transcription
termination
region
Downstream
Poly-A
signal
Intron
Transcription
Cleaved
3 end of
primary
transcript
5
Primary RNA
transcript
(pre-mRNA)
Intron RNA
RNA processing
mRNA
Coding segment
5 Cap 5 UTR
Start
codon
Stop
codon 3 UTR
3
Poly-A
tail
P
P
P
G AAA AAA

The Roles of Transcription Factors
• To initiate transcription, eukaryotic RNA
polymerase requires the assistance of proteins
called transcription factors
• General transcription factors are essential for the
transcription of all protein-coding genes
• In eukaryotes, high levels of transcription of
particular genes depend on control elements
interacting with specific transcription factors

• Proximal control elements are located close to the
promoter
• Distal control elements, groupings of which are
called enhancers, may be far away from a gene
or even located in an intron
Enhancers and Specific Transcription Factors

• An activator is a protein that binds to an enhancer
and stimulates transcription of a gene
• Activators have two domains, one that binds DNA
and a second that activates transcription
• Bound activators facilitate a sequence of protein-
protein interactions that result in transcription of a
given gene

Figure 18.9
DNA
Activation
domain
DNA-binding
domain

• Some transcription factors function as repressors,
inhibiting expression of a particular gene by a
variety of methods
• Some activators and repressors act indirectly by
influencing chromatin structure to promote or
silence transcription

Activators
DNA
Enhancer
Distal control
element
Promoter
Gene
TATA box
Figure 18.10-1

Activators
DNA
Enhancer
Distal control
element
Promoter
Gene
TATA box
General
transcription
factors
DNA-
bending
protein
Group of mediator proteins
Figure 18.10-2

Activators
DNA
Enhancer
Distal control
element
Promoter
Gene
TATA box
General
transcription
factors
DNA-
bending
protein
Group of mediator proteins
RNA
polymerase II
RNA
polymerase II
RNA synthesis
Transcription
initiation complex
Figure 18.10-3

Figure 18.11
Control
elements
Enhancer Promoter
Albumin gene
Crystallin
gene
LIVER CELL
NUCLEUS
Available
activators
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
LENS CELL
NUCLEUS
Available
activators
Albumin gene
not expressed
Crystallin gene
expressed
(b) Lens cell

Control
elements
Enhancer Promoter
Albumin gene
Crystallin
gene
LIVER CELL
NUCLEUS
Available
activators
Albumin gene
expressed
Crystallin gene
not expressed
(a) Liver cell
Figure 18.11a

Control
elements
Enhancer Promoter
Albumin gene
Crystallin
gene
LENS CELL
NUCLEUS
Available
activators
Albumin gene
not expressed
Crystallin gene
expressed
(b) Lens cell
Figure 18.11b

Coordinately Controlled Genes in
Eukaryotes
• Unlike the genes of a prokaryotic operon, each of
the co-expressed eukaryotic genes has a
promoter and control elements
• These genes can be scattered over different
chromosomes, but each has the same
combination of control elements
• Copies of the activators recognize specific control
elements and promote simultaneous transcription
of the genes

Nuclear Architecture and Gene Expression
• Loops of chromatin extend from individual
chromosomes into specific sites in the nucleus
• Loops from different chromosomes may
congregate at particular sites, some of which are
rich in transcription factors and RNA polymerases
• These may be areas specialized for a common
function

Figure 18.12
Chromosome
territory
Chromosomes in the
interphase nucleus
Chromatin
loop
Transcription
factory
10 m

Figure 18.12a
Chromosomes in the
interphase nucleus
10 m

Mechanisms of Post-Transcriptional
Regulation
• Transcription alone does not account for gene
expression
• Regulatory mechanisms can operate at various
stages after transcription
• Such mechanisms allow a cell to fine-tune gene
expression rapidly in response to environmental
changes

RNA Processing
• In alternative RNA splicing, different mRNA
molecules are produced from the same primary
transcript, depending on which RNA segments are
treated as exons and which as introns

Exons
DNA
Troponin T gene
Primary
RNA
transcript
RNA splicing
or
mRNA
1
1
1 1
2
2
2 2
3
3
3
4
4
4
5
5
5 5
Figure 18.13

mRNA Degradation
• The life span of mRNA molecules in the cytoplasm
is a key to determining protein synthesis
• Eukaryotic mRNA is more long lived than
prokaryotic mRNA
• Nucleotide sequences that influence the lifespan
of mRNA in eukaryotes reside in the untranslated
region (UTR) at the 3 end of the molecule

Initiation of Translation
• The initiation of translation of selected
mRNAs can be blocked by regulatory proteins that
bind to sequences or structures of the mRNA
• Alternatively, translation of all mRNAs
in a cell may be regulated simultaneously
• For example, translation initiation factors are
simultaneously activated in an egg following
fertilization

Protein Processing and Degradation
• After translation, various types of protein
processing, including cleavage and the addition of
chemical groups, are subject to control
• Proteasomes are giant protein complexes that
bind protein molecules and degrade them

Figure 18.14
Protein to
be degraded
Ubiquitin
Ubiquitinated
protein
Proteasome
Protein entering
a proteasome
Proteasome
and ubiquitin
to be recycled
Protein
fragments
(peptides)

Noncoding RNAs play multiple roles in
controlling gene expression
• Only a small fraction of DNA codes for proteins,
and a very small fraction of the non-protein-coding
DNA consists of genes for RNA such as rRNA and
tRNA
• A significant amount of the genome may be
transcribed into noncoding RNAs (ncRNAs)
• Noncoding RNAs regulate gene expression at two
points: mRNA translation and chromatin
configuration

Effects on mRNAs by MicroRNAs and
Small Interfering RNAs
• MicroRNAs (miRNAs) are small single-stranded
RNA molecules that can bind to mRNA
• These can degrade mRNA or block its translation

(a) Primary miRNA transcript
Hairpin
miRNA
miRNA
Hydrogen
bond
Dicer
miRNA-
protein
complex
mRNA degraded Translation blocked
(b) Generation and function of miRNAs
5 3
Figure 18.15

• The phenomenon of inhibition of gene expression
by RNA molecules is called RNA interference
(RNAi)
• RNAi is caused by small interfering RNAs
(siRNAs)
• siRNAs and miRNAs are similar but form from
different RNA precursors

Chromatin Remodeling and Effects on
Transcription by ncRNAs
• In some yeasts siRNAs play a role in
heterochromatin formation and can block large
regions of the chromosome
• Small ncRNAs called piwi-associated RNAs
(piRNAs) induce heterochromatin, blocking the
expression of parasitic DNA elements in the
genome, known as transposons
• RNA-based mechanisms may also block
transcription of single genes

The Evolutionary Significance of Small
ncRNAs
• Small ncRNAs can regulate gene expression at
multiple steps
• An increase in the number of miRNAs in a species
may have allowed morphological complexity to
increase over evolutionary time
• siRNAs may have evolved first, followed by
miRNAs and later piRNAs

A program of differential gene
expression leads to the different cell
types in a multicellular organism
• During embryonic development, a fertilized egg
gives rise to many different cell types
• Cell types are organized successively into tissues,
organs, organ systems, and the whole organism
• Gene expression orchestrates the developmental
programs of animals

A Genetic Program for Embryonic
Development
• The transformation from zygote to adult results
from cell division, cell differentiation, and
morphogenesis

Figure 18.16
(a) Fertilized eggs of a frog (b) Newly hatched tadpole
1 mm 2 mm

Figure 18.16a
(a) Fertilized eggs of a frog
1 mm

Figure 18.16b
(b) Newly hatched tadpole
2 mm

• Cell differentiation is the process by which cells
become specialized in structure and function
• The physical processes that give an organism its
shape constitute morphogenesis
• Differential gene expression results from genes
being regulated differently in each cell type
• Materials in the egg can set up gene regulation
that is carried out as cells divide

Cytoplasmic Determinants and
Inductive Signals
• An egg’s cytoplasm contains RNA, proteins, and
other substances that are distributed unevenly in
the unfertilized egg
• Cytoplasmic determinants are maternal
substances in the egg that influence early
development
• As the zygote divides by mitosis, cells contain
different cytoplasmic determinants, which lead to
different gene expression

Figure 18.17
(a) Cytoplasmic determinants in the egg (b) Induction by nearby cells
Unfertilized egg
Sperm
Fertilization
Zygote
(fertilized egg)
Mitotic
cell division
Two-celled
embryo
Nucleus
Molecules of two
different cytoplasmic
determinants
Early embryo
(32 cells)
NUCLEUS
Signal
transduction
pathway
Signal
receptor
Signaling
molecule
(inducer)

Figure 18.17a
(a) Cytoplasmic determinants in the egg
Unfertilized egg
Sperm
Fertilization
Zygote
(fertilized egg)
Mitotic
cell division
Two-celled
embryo
Nucleus
Molecules of two
different cytoplasmic
determinants

• The other important source of developmental
information is the environment around the cell,
especially signals from nearby embryonic cells
• In the process called induction, signal molecules
from embryonic cells cause transcriptional
changes in nearby target cells
• Thus, interactions between cells induce
differentiation of specialized cell types

Figure 18.17b
(b) Induction by nearby cells
Early embryo
(32 cells)
NUCLEUS
Signal
transduction
pathway
Signal
receptor
Signaling
molecule
(inducer)

Sequential Regulation of Gene
Expression During Cellular
Differentiation
• Determination commits a cell to its final fate
• Determination precedes differentiation
• Cell differentiation is marked by the production of
tissue-specific proteins

• Myoblasts produce muscle-specific proteins and
form skeletal muscle cells
• MyoD is one of several “master regulatory genes”
that produce proteins that commit the cell to
becoming skeletal muscle
• The MyoD protein is a transcription factor that
binds to enhancers of various target genes

Nucleus
Embryonic
precursor cell
DNA
Master regulatory
gene myoD
OFF OFF
Other muscle-specific genes
Figure 18.18-1

Nucleus
Embryonic
precursor cell
Myoblast
(determined)
DNA
Master regulatory
gene myoD
OFF OFF
OFF
mRNA
MyoD protein
(transcription
factor)
Figure 18.18-2

Nucleus
Embryonic
precursor cell
Myoblast
(determined)
Part of a muscle fiber
(fully differentiated cell)
DNA
Master regulatory
gene myoD
OFF OFF
OFF
mRNA
MyoD protein
(transcription
factor)
mRNA mRNA mRNA mRNA
MyoD Another
transcription
factor
Myosin, other
muscle proteins,
and cell cycle–
blocking proteins
Figure 18.18-3

Pattern Formation: Setting Up the Body
Plan
• Pattern formation is the development of a spatial
organization of tissues and organs
• In animals, pattern formation begins with the
establishment of the major axes
• Positional information, the molecular cues that
control pattern formation, tells a cell its location
relative to the body axes and to neighboring cells

• Pattern formation has been extensively studied in
the fruit fly Drosophila melanogaster
• Combining anatomical, genetic, and biochemical
approaches, researchers have discovered
developmental principles common to many other
species, including humans

The Life Cycle of Drosophila
• In Drosophila, cytoplasmic determinants in the
unfertilized egg determine the axes before
fertilization
• After fertilization, the embryo develops into a
segmented larva with three larval stages

Head Thorax Abdomen
0.5 mm
BODY
AXES
Anterior
Left
Ventral
Dorsal
Right
Posterior
(a) Adult
Egg
developing within
ovarian follicle
Follicle cell
Nucleus
Nurse cell
Egg
Unfertilized egg
Depleted
nurse cells
Egg
shell
Fertilization
Laying of egg
Fertilized egg
Embryonic
development
Segmented
embryo
Body
segments
Hatching
0.1 mm
Larval stage
(b) Development from egg to larva
2
1
3
4
5
Figure 18.19

Figure 18.19a
Head Thorax Abdomen
0.5 mm
BODY
AXES
Anterior
Left
Ventral
Dorsal
Right
Posterior
(a) Adult

Figure 18.19b
Egg
developing within
ovarian follicle
Follicle cell
Nucleus
Nurse cell
Egg
Unfertilized egg
Depleted
nurse cells
Egg
shell
Fertilization
Laying of egg
Fertilized egg
Embryonic
development
Segmented
embryo
Body segments Hatching
0.1 mm
Larval stage
(b) Development from egg to larva
5
4
3
2
1

Genetic Analysis of Early Development:
Scientific Inquiry
• Edward B. Lewis, Christiane Nüsslein-Volhard,
and Eric Wieschaus won a Nobel Prize in 1995
for decoding pattern formation in Drosophila
• Lewis discovered the homeotic genes, which
control pattern formation in late embryo, larva,
and adult stages

Figure 18.20
Wild type Mutant
Eye
Antenna
Leg

Figure 18.20a
Wild type
Eye
Antenna

• Nüsslein-Volhard and Wieschaus studied segment
formation
• They created mutants, conducted breeding
experiments, and looked for corresponding genes
• Many of the identified mutations were embryonic
lethals, causing death during embryogenesis
• They found 120 genes essential for normal
segmentation

Axis Establishment
• Maternal effect genes encode for cytoplasmic
determinants that initially establish the axes of the
body of Drosophila
• These maternal effect genes are also called egg-
polarity genes because they control orientation of
the egg and consequently the fly

• One maternal effect gene, the bicoid gene, affects
the front half of the body
• An embryo whose mother has no functional bicoid
gene lacks the front half of its body and has
duplicate posterior structures at both ends
Bicoid: A Morphogen Determining Head
Structures

Figure 18.21
Head Tail
Tail Tail
Wild-type larva
Mutant larva (bicoid)
250 m
T1 T2
T3
A1 A2 A3 A4 A5
A6
A7
A8
A8
A7
A6
A7
A8

Figure 18.21a
Head Tail
Wild-type larva 250 m
T1 T2
T3
A1 A2 A3 A4 A5
A6
A7
A8

Figure 18.21b
Tail Tail
Mutant larva (bicoid)
A8
A7
A6
A7
A8

• This phenotype suggests that the product of the
mother’s bicoid gene is concentrated at the future
anterior end
• This hypothesis is an example of the morphogen
gradient hypothesis, in which gradients of
substances called morphogens establish an
embryo’s axes and other features

Figure 18.22
Bicoid mRNA in mature
unfertilized egg
Bicoid mRNA in mature
unfertilized egg
Fertilization,
translation of
bicoid mRNA
Anterior end
100 m
Bicoid protein in
early embryo
Bicoid protein in
early embryo
RESULTS

• The bicoid research is important for three reasons
– It identified a specific protein required for some
early steps in pattern formation
– It increased understanding of the mother’s role in
embryo development
– It demonstrated a key developmental principle that
a gradient of molecules can determine polarity
and position in the embryo

Cancer results from genetic changes
that affect cell cycle control
• The gene regulation systems that go wrong during
cancer are the very same systems involved in
embryonic development

Types of Genes Associated with Cancer
• Cancer can be caused by mutations to genes that
regulate cell growth and division
• Tumor viruses can cause cancer in animals
including humans

• Oncogenes are cancer-causing genes
• Proto-oncogenes are the corresponding normal
cellular genes that are responsible for normal cell
growth and division
• Conversion of a proto-oncogene to an oncogene
can lead to abnormal stimulation of the cell cycle

Figure 18.23
Proto-oncogene
DNA
Translocation or
transposition: gene
moved to new locus,
under new controls
Gene amplification:
multiple copies of
the gene
New
promoter
Normal growth-
stimulating
protein in excess
Normal growth-stimulating
protein in excess
Point mutation:
within a control
element
within
the gene
Oncogene Oncogene
Normal growth-
stimulating
protein in
excess
Hyperactive or
degradation-
resistant
protein

• Proto-oncogenes can be converted to oncogenes
by
– Movement of DNA within the genome: if it ends up
near an active promoter, transcription may
increase
– Amplification of a proto-oncogene: increases the
number of copies of the gene
– Point mutations in the proto-oncogene or its
control elements: cause an increase in gene
expression

Tumor-Suppressor Genes
• Tumor-suppressor genes help prevent
uncontrolled cell growth
• Mutations that decrease protein products of tumor-
suppressor genes may contribute to cancer onset
• Tumor-suppressor proteins
– Repair damaged DNA
– Control cell adhesion
– Inhibit the cell cycle in the cell-signaling pathway

Interference with Normal Cell-Signaling
Pathways
• Mutations in the ras proto-oncogene and p53
tumor-suppressor gene are common in human
cancers
• Mutations in the ras gene can lead to production
of a hyperactive Ras protein and increased cell
division

Figure 18.24
Growth
factor
1
2
3
4
5
1
2
Receptor
G protein
Protein kinases
(phosphorylation
cascade)
NUCLEUS
Transcription
factor (activator)
DNA
Gene expression
Protein that
stimulates
the cell cycle
Hyperactive Ras protein
(product of oncogene)
issues signals on its
own.
(a) Cell cycle–stimulating pathway
MUTATION
Ras
Ras
GTP
GTP
P
P
P P
P
P
(b) Cell cycle–inhibiting pathway
Protein kinases
UV
light
DNA damage
in genome
Active
form
of p53
DNA
Protein that
inhibits
the cell cycle
Defective or missing
transcription factor,
such as
p53, cannot
activate
transcription.
MUTATION
EFFECTS OF MUTATIONS
(c) Effects of mutations
Protein
overexpressed
Cell cycle
overstimulated
Increased cell
division
Protein absent
Cell cycle not
inhibited
3

Growth
factor
1
Receptor
G protein
Protein kinases
(phosphorylation
cascade)
NUCLEUS
Transcription
factor (activator)
DNA
Gene expression
Protein that
stimulates
the cell cycle
Hyperactive Ras protein
(product of oncogene)
issues signals on its
own.
(a) Cell cycle–stimulating pathway
MUTATION
Ras
GTP
P
P
P
P
P
P
2
3
4
5
Ras
GTP
Figure 18.24a

Figure 18.24b
(b) Cell cycle–inhibiting pathway
Protein kinases
UV
light
DNA damage
in genome
Active
form
of p53
DNA
Protein that
inhibits
the cell cycle
Defective or missing
transcription factor,
such as
p53, cannot
activate
transcription.
MUTATION
2
1
3

• Suppression of the cell cycle can be important in
the case of damage to a cell’s DNA; p53 prevents
a cell from passing on mutations due to DNA
damage
• Mutations in the p53 gene prevent suppression of
the cell cycle

Figure 18.24c
EFFECTS OF MUTATIONS
(c) Effects of mutations
Protein
overexpressed
Cell cycle
overstimulated
Increased cell
division
Protein absent
Cell cycle not
inhibited

The Multistep Model of Cancer
Development
• Multiple mutations are generally needed for full-
fledged cancer; thus the incidence increases with
age
• At the DNA level, a cancerous cell is usually
characterized by at least one active oncogene and
the mutation of several tumor-suppressor genes

Figure 18.25
Colon
Normal colon
epithelial cells
Loss
of tumor-
suppressor
gene APC
(or other)
1
2
3
4
5
Colon wall
Small benign
growth
(polyp)
Activation
of ras
oncogene
Loss
of tumor-
suppressor
gene DCC
Loss
of tumor-
suppressor
gene p53
Additional
mutations
Malignant
tumor
(carcinoma)
Larger
benign growth
(adenoma)

Inherited Predisposition and Other
Factors Contributing to Cancer
• Individuals can inherit oncogenes or mutant alleles
of tumor-suppressor genes
• Inherited mutations in the tumor-suppressor gene
adenomatous polyposis coli are common in
individuals with colorectal cancer
• Mutations in the BRCA1 or BRCA2 gene are found
in at least half of inherited breast cancers, and
tests using DNA sequencing can detect these
mutations

Biotechnology

Overview: The DNA Toolbox
• Sequencing of the genomes of more than 7,000
species was under way in 2010
• DNA sequencing has depended on advances in
technology, starting with making recombinant DNA
• In recombinant DNA, nucleotide sequences from
two different sources, often two species, are
combined in vitro into the same DNA molecule

• Methods for making recombinant DNA are central
to genetic engineering, the direct manipulation of
genes for practical purposes
• DNA technology has revolutionized
biotechnology, the manipulation of organisms or
their genetic components to make useful products
• An example of DNA technology is the microarray,
a measurement of gene expression of thousands
of different genes

Molecular biology for post graduates.ppt

Molecular biology for post graduates.ppt

Recommended

Recommended

More Related Content

Similar to Molecular biology for post graduates.ppt

Similar to Molecular biology for post graduates.ppt (20)

More from AhmedKasem39

More from AhmedKasem39 (6)

Recently uploaded

Recently uploaded (20)

Molecular biology for post graduates.ppt