Genetika Mikroba - Tugas 24 Nov 2016 PDF

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A mating dance
between cells connected
by conjugation pili.
Microbial
Genetics
Plasmida circular
snippet of DNA carrying
genetic treasures.
Acinetobactertiny
nonmotile gramnegative bacilli, coming
to a hospital in your
neighborhood?
Over time, so many soldiers suffered battlefieldrelated infections by this ubiquitous pathogen that
it was given the nickname Iraqnobacter.
CASE FILE
At War with an Emerging Pathogen
55-year-old woman being treated

in intensive care for a lung transplant developed a case of pneumonia from a ventilator she needed for
breathing. The bacteria isolated from her
lungs turned out to be Acinetobacter baumannii, a widespread resident of soil and
water that has found its way into hospitals and long-term care facilities. Tests
showed that all of the drugs available to
treat the infection were ineffective against
the pathogenexcept for colistin. Early
in the treatment with this drug, the lung
infection appeared to be clearing up, but
after a few days, it returned and the patient died of respiratory failure. Cultures
taken late in the case detected a new
strain of the infectious agent that had developed resistance to all known drugs,
including colistin.
During the same period, several other
patients in the ICU developed drugresistant A. baumannii infections. This
sudden outbreak of nosocomial infections
spurred an investigation by an infection
control nurse, who took samples from
254
multiple locations and cultured them. The

bacteria were isolated from nearly every
possible source, including medical devices, patient skin samples, bedding, hospital workers, the air, and room surfaces.
A world away in the Iraq and Afghanistan wars, this same microbe became
one of the most fearsome pathogens to
infect soldiers with battle injuries. In one
case, a 55-year-old man sustained a grenade injury while transporting soldiers.
Shrapnel penetrated his thigh and fractured the femur. When X rays revealed
the presence of an infection in his broken
bone, he was transported to a trauma
hospital. Cultures of the wound isolated a
gram-negative coccobacillus identied as
Acinetobacter baumannii. Testing indicated that it was resistant to all drugs
except for an experimental drug, tigecycline. After a several weeks course of
tigecycline, the infection was resolved and
the patient was released. Over time, so
many soldiers suffered battleeld-related
infections by this ubiquitous pathogen that
it was given the nickname Iraqnobacter.
For many years, the rise of superbugs has become a major concern of the
medical community. The working denition of this term includes any bacterial
species that have developed signicant
resistance to several drugs that would ordinarily have been given in therapy. Two
of the A. baumannii strains in these cases
are dened as multiple drug resistant
(MDR), in that they resist a fairly broad
range of drugs. One strain ts the category of extreme drug resistance (XDR),
meaning that it is resistant to all possible
drugs. Both versions of this pathogen
have been added to the list of emerging
diseases.
What is the probable origin of the

pathogen, and what is meant by the
term nosocomial?
What does drug resistance mean from

a genetic standpoint?
To continue the case, go to page 285.
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9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity
9.1 Introduction to Genetics and Genes:

Unlocking the Secrets of Heredity
xpected Learning Outcomes
1. Dene heredity, genetics, genome, gene, phenotype, and

genotype.
2. Discuss the basic nature of genetic material in eukaryotes,

prokaryotes, and viruses.
3. Explain how DNA is organized and packaged.
4. Describe the chemical structure of DNA and its signicance.
5. List the nitrogen bases and explain their bonding patterns.
6. Describe the process of DNA replication as it occurs in
prokaryotic cells.
Genetics is the science that studies the inheritance of biological

characteristics by living things. This subject, also known as heredity, is a wide-ranging science that examines:

the transmission of biological properties (traits) from parent to

offspring,
the expression and variation of those traits,
the structure and function of the genetic material, and
how this material changes or evolves.
The study of genetics takes place on several levels (figure 9.1).

Organismal genetics observes the transmission and expression of
genetic factors in the whole organism or cell; chromosomal genetics examines the characteristics and actions of chromosomes; and
molecular genetics deals with the biochemistry of gene function.
All of these levels provide insight into microbial structure, physiology, evolution, and pathogenicity. It should also be noted that the
study of microbial genetics will emphasize universal themes in the
genetics of all organisms, and it will increase appreciation for the
astounding advances in genetic engineering covered in chapter 10.
Organism level
The Nature of the Genetic Material

Deoxyribonucleic acid or DNA is the central molecule of genetics,
so it is essential that you develop a working knowledge of its characteristics to understand a broad range of genetic principles. With this
in mind, we will use the first section of this chapter to lay some
groundwork on DNA organization, structure, and duplication. This
will be followed by coverage of related concepts such as RNA and
protein synthesis, genetic control, mutations, and genetic transfers
in the remaining five sections.
The Levels of Structure and

Function of the Genome
The genome is the sum total of genetic material carried within a
cell. Although most of the genome exists in the form of chromosomes, genetic material can appear in nonchromosomal sites as
well (figure 9.2). For example, bacteria and some fungi contain tiny
extra pieces ofDNA (plasmids), and the mitochondria and chloroplasts of eukaryotes are equipped with their own functional chromosomes. Genomes of cells are composed exclusively of DNA, but
viruses contain either DNA or RNA as the principal genetic material. Although the specific genome of an individual organism is
unique, the general pattern of nucleic acid structure and function is
similar among all organisms.
In general, a chromosome is a discrete cellular structure composed of a neatly packaged DNA molecule. The chromosomes of
eukaryotes and bacterial cells differ in several respects. The structure of eukaryotic chromosomes consists of a DNA molecule
tightly wound around histone proteins, whereas a bacterial chromosome is condensed and secured into a packet by means of a
different type of protein. Eukaryotic chromosomes are located in
the nucleus; they vary in number from a few to hundreds; they can
occur in pairs (diploid) or singles (haploid); and they are linear in
format. In contrast, most bacteria have a single, circular chromosome, although some have multiple chromosomes and a few have
linear chromosomes.
Cell level
Chromosome level
Molecular level
TA
GC
AT
C
CG
TA
GC
A
TA
GC
CG
A
TA
CG
A
T
GC
CG
Figure 9.1 Levels of genetic study. The operations of genetics can be observed at the levels of organism, cell, chromosome, and DNA
sequence (molecular level). Although levels shown here are for a eukaryotic organism they would follow a similar pattern in prokaryotes.
256
Chapter 9 Microbial Genetics
Cells
Prokaryote
Eukaryote
(composite)
Chromosomes
Nucleus
Nucleolus
Chromosome
Plasmids
Mitochondrion
Plasmid
(in some
fungi and
protozoa)
Viruses
Extrachromosomal
DNA
Chloroplast
Figure 9.2
DNA
RNA
The general location and forms of the genome in two cell types and selected viruses (not to scale).
All chromosomes contain a series of basic informational

packets called genes. A gene can be defined from more than one
perspective. In classical genetics, the term refers to the fundamental
unit of heredity responsible for a given trait in an organism. In the
molecular and biochemical sense, it is a portion of the chromosome
that provides information for a given cell function. More specifically still, it is a specific segment of DNA that contains the necessary codes to make a protein or RNA molecule. This last definition
of a gene is emphasized in this chapter.
Genes fall into three basic categories: structural genes that code
for proteins, genes that code for RNA, and regulatory genes that
control gene expression. The sum of all of these types of genes constitutes an organisms distinctive genetic makeup, or genotype.*
The expression of the genotype creates traits (certain structures or
functions) referred to as the phenotype.* For example a person inherits a combination of genes (genotype) that gives a certain eye
color or height (phenotype), a bacterium inherits genes that direct
the formation of a flagellum or the ability to metabolize a certain
substrate, and a virus has genes that dictate its capsid structure. All
organisms contain more genes in their genotypes than are being seen
as a phenotype at any given time. In other words, the phenotype can
change depending on which genes are turned on or expressed.
across, making the stretched-out DNA 1,000 times longer than the
cell (figure 9.3). Still, the bacterial chromosome takes up only
about one-third to one-half of the cells volume. Likewise, if the
sum of all DNA contained in the 46 human chromosomes were
unraveled and laid end to end, it would measure about 6 feet. How
can such elongated genomes fit into the minuscule volume of a cell
and, in the case of eukaryotes, into an even smaller compartment,
the nucleus? The answer lies in the complex coiling of the DNA
chain (Insight 9.1).
The Structure of DNA: A Double Helix

with Its Own Language
To analyze the structure of DNA at the molecular level, imagine
being able to magnify a small piece of a gene about 5 million
The Size and Packaging of Genomes

Genomes vary greatly in size. Viruses have anywhere from a few to
several hundred genes; the bacterium Escherichia coli has a single
chromosome containing 4,288 genes, and a human cell packs about
five times that many into 46 chromosomes. The chromosome of
E. coli would measure about 1 mm if unwound and stretched out
linearly, and yet this fits within a cell that measures just over 1 m
* genotype (jee9-noh-typ) Gr. gennan, to produce, and typos, type.

* phenotype (fee9-noh-typ) Gr. phainein, to show. The physical manifestation of
gene expression.
Figure 9.3 A disrupted Escherichia colicell has spewed out its

chromosome in a single, uncoiled DNA strand.
257
INSIGHT 9.1
The Packaging of DNA: Winding, Twisting, and Coiling
Packing the mass of DNA into the cell involves compacting the DNA
molecule by means of supercoils or superhelices. In the simpler system
of prokaryotes, the circular chromosome is packaged by the action of a
special enzyme called a topoisomerase (specifically, DNA gyrase). This
enzyme coils the chromosome into a tight bundle by introducing a reversible series of twists into the DNA molecule.
The system in eukaryotes is more complex, with three or more
levels of coiling. First, the DNA molecule of a chromosome, which is
linear, is wound twice around the histone proteins, creating a chain of
nucleosomes. The nucleosomes fold in a spiral formation upon one another. An even greater supercoiling occurs when this spiral arrangement
further twists on its radius into a giant spiral with loops radiating from
the outside. This extreme degree of compactness is what makes the
eukaryotic chromosome visible during mitosis.
The condensation of DNA and protein into chromatin was once

thought to function mainly as a way for such a long molecule to fit into a
cell. However, new research has shown that the coiling of DNA also
serves to make certain segments of the genetic program either more or
less available to the cell. This is a major control on the expression of
many traits, especially in eukaryotes. Having an additional regulatory
function creates an entirely new set of information to guide phenotypic
expression. The realization that the phenotype of a cell is due to more
than just the order of nucleotides in the DNA has led to a rethinking of
some of the most basic aspects of genetics (see Insight 9.3).
Because genetic codes cannot be properly accessed when DNA is
supercoiled, what other system would a cell need to manage
DNA? Answer available at http://www.mhhe.com/talaro8
DNA
DNA
double helix
Condensed metaphase
chromosome
Chemical tags attached to histone

proteins may increase the
expression of nearby genes.
DNA wrapped around

histones with linker
DNA between them
DNA
Histone
Nucleosome
Supercoiled condensed
chromatin
Condensed
nucleosomes
Loosely condensed
chromosome
Uncondensed
chromatin fiber
Chromatin
The packaging of DNA. Eukaryotic DNA is intimately

associated with a variety of proteins and small molecules.
This relationship modulates the expression of many genes
while at the same time allowing for the ordered
condensation of DNA into a compact molecule.
Backbone
times. What you would see is one of the great marvels of life. The
pieces of this molecular puzzle were finally worked out by James
Watson and Francis Crick in 1953 (Insight 9.2). They discovered
that DNA is a giant molecule with two strands forming a double
helix. Extensive evidence shows that the general structure of
DNA is universal, except in some viruses that contain singlestranded DNA.
The basic unit of DNA structure is a nucleotide, composed of
phosphate, deoxyribose sugar, and a nitrogen base, as shown in
this simplified model (see figure 2.23):
Deoxyribose
sugar
N base
D
Hydrogen
bonds
P
D
Phosphate
DNA
P
G
P
D
258
INSIGHT 9.2
Deciphering the Structure of DNA
The search for the primary molecules of heredity was a serious focus throughout the first half
of the twentieth century. At first, many biologists thought that protein was the genetic material. An important milestone occurred in 1944
when Oswald Avery, Colin MacLeod, and
Maclyn McCarty purified DNA and demonstrated at last that it was indeed the blueprint
for life. This was followed by an avalanche of
research, which continues today.
X rays produce a photographic image that can

predict the three-dimensional structure of the
molecule. After being allowed to view certain
X-ray data, Watson and Crick noticed an unmistakable pattern: The molecule appeared to
be a double helix. Gradually, the pieces of the
puzzle fell into place, and a final model was
assembleda model that explained all of the
qualities of DNA, including how it is copied.
Although Watson and Crick were rightly hailed
for the clarity of their solution, it must be emWe Have Discovered the
phasized that their success was due to the conSecret of Life.
siderable efforts of a number of English and
American scientists. This historic discovery
One area of extreme interest concerned the
showed that the tools of physics and chemistry
molecular structure of DNA. In 1951, American
have useful applications in biological systems,
biologist James Watson and English physicist
and it also spawned ingenious research in all
Francis Crick collaborated on solving the DNA
areas of molecular genetics.
puzzle. Although they did little of the original
Since the discovery of the double helix in
research, they were intrigued by several findings
1953, an extensive body of biochemical, microfrom other scientists. It had been determined by
The men who cracked the code of life.
scopic, and crystallographic analysis has left
Erwin Chargaff that any model of DNA structure
Dr. James Watson (left) and Dr. Francis Crick
little doubt that the model first proposed by
would have to contain deoxyribose, phosphate,
(right) stand next to their model that nally
Watson and Crick is correct. Scanning tunnelpurines, and pyrimidines arranged in a way that
explained the structure of DNA in 1953.
ing microscopes produce three-dimensional
would provide variation and a simple way of
images
of
DNA
that
verify
the helical shape and twists of DNA reprecopying itself. Watson and Crick spent long hours constructing models
sented by models.
with cardboard cutouts and kept alert for any and every bit of information
that might give them an edge.
What particular requirements did Watson and Cricks model of
Two English biophysicists, Maurice Wilkins and Rosalind Franklin,
DNA have to fit to be accurate? Answer available at http://www.
had been painstakingly collecting data on X-ray crystallographs of DNA
mhhe.com/talaro8
for several years. With this technique, molecules of DNA bombarded by
Each deoxyribose sugar bonds covalently in a repeating pattern with two phosphates. One of the bonds is to the number 59
(read five prime) carbon on deoxyribose, and the other is to the
39 carbon, which specifies the order and direction of each strand
(figure 9.4). This formation results in an elongate strand with a
sugar-phosphate backbone.
The nitrogen bases, purines and pyrimidines, attach by covalent bonds at the 19 position of the sugar (figure 9.4a). They span
the center of the molecule and pair with appropriate complementary bases from the other strand, thereby forming a double-stranded
helix. The paired bases are so aligned as to be joined by hydrogen
bonds. Such weak bonds are easily broken, allowing the molecule
to be unzipped into its complementary strands. This feature is of
great importance in gaining access to the information encoded in
the nitrogen base sequence. Pairing of purines and pyrimidines is
not random; it is dictated by the formation of hydrogen bonds between certain bases. Thus, in DNA, the purine adenine (A) pairs
with the pyrimidine thymine (T), and the purine guanine (G) pairs
with the pyrimidine cytosine (C). Note that adenine forms two
hydrogen bonds with thymine, and cytosine forms three hydrogen
bonds with guanine. This difference will influence a number of

DNA functions. New research also indicates that the bases are attracted to each other in this pattern because each has a complementary three-dimensional shape that matches its pair. Although the
base-pairing partners generally do not vary, the sequence of base
pairs along the DNA molecule can assume any order, resulting in
an infinite number of possible nucleotide sequences.
Other important considerations of DNA structure concern the
nature of the double helix itself. The two strands are not oriented in
the same direction. One side of the helix runs in the opposite direction of the other, in what is called an antiparallel arrangement
(figure 9.4b). The order of the bond between the carbon on deoxyribose and the phosphates is used to keep track of the direction of
the two sides of the helix. Thus, one helix runs from the 59 to 39
direction, and the other runs from the 39 to 59 direction. This characteristic is a significant factor in DNA synthesis and translation.
As apparently perfect and regular as the DNA molecule may seem,
it is not exactly symmetrical. The torsion in the helix and the stepwise stacking of the nitrogen bases produce two different-size surface features, the major and minor grooves (figure 9.4c).
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H
N
H N
G N H
N C
H
N
N H
Sugar
H
Sugar
phosphate
Nitrogen
base pairs
Sugar
phosphate
5
3
OH
5
4 D
3 2
D
C
D
P
Phosphate
5
4
P
P
Base pairs
Sugar phosphate
backbone
D 1 Deoxyribose
with carbon number
2
C
Cytosine
Guanine
Thymine
Adenine
Minor
groove
P
A
5
D
Covalent
bond
Major
groove
Hydrogen
bond
(b)
(c)
OH
H
N
N H
A N
O
H N T
N
Sugar
(a)
CH3
H
N
Figure 9.4 Three views of DNA structure. (a) A schematic nonhelical model shows
the basic layout of the two strands. Note that the order of phosphate and sugar bonds goes
in the opposite direction for the two strands, going from the 59 carbon to the 39 carbon on
one strand and from the 39 carbon to the 59 carbon on the other strand. Insets show details
of the nitrogen base pairs. (b) Simplied model that highlights the antiparallel arrangement
and the major and minor grooves. (c) Space-lling model that more accurately depicts the
three-dimensional structure of DNA.
One of the significant findings that has emerged from sequencing

the DNA of chromosomes is knowing how long it can be. An average
bacterial chromosome consists of 5 million to 6 million nucleotides;
the 46 chromosomes of humans amount to about 3 billion nucleotides.
DNA Replication: Preserving the Code

and Passing It On
For a species to survive, it must reproduce. In bacteria, this involves
division of the cell by means of binary fission or budding, but it also
involves the accurate duplication and separation of the genetic material into each daughter cell to ensure normal function.
It is the sequence of bases along the length of DNA that constitutes its language. For this language to be preserved for hundreds of generations, the codes it contains will need to be
duplicated with high fidelity. This process of duplication is called
DNA replication. In the following example, we will show replication in bacteria, but with some exceptions, it also applies to the
process as it works in eukaryotes and some viruses. Recall from
chapter 7 that early in binary fission, the metabolic machinery of a
259
260
bacterium is programmed to initiate the duplication of the chromosome. Despite its complexity, DNA replication can be very rapid:
This must be completed in a single generation time (around
20minutes in E. coli).
G
C
T A
TA
GC
AT
A
T A
The arrangement of nitrogen bases in DNA has two essential

effects.
It is tempting to consider how such a seemingly simple

set of codes can account for the extreme differences among
forms as diverse as a virus, E. coli, and a human. The English
language, based on 26 letters, can create an innite variety of
words, but how can an apparently complex genetic language
such as DNA be based on just four nitrogen base letters?
The answer lies in the fact that the four bases of DNA are arranged into much longer sequences. An average bacterial
gene may contain 1,000 nucleotides: so while DNA contains
fewer letters, they are arranged into much longer words.
Mathematically speaking, 1,000 nucleotides can be arranged
in 41,000 different sequences, a number so large (1.5 3 10602)
that it provides nearly endless degrees of variation.
The Overall Replication Process

What features allow the DNA molecule to be exactly duplicated,
and how is its integrity retained? DNA replication requires a careful
orchestration of the actions of 30 different enzymes (partial list in
table 9.1), which separate the strands of the existing DNA molecule,
copy its template, and produce two complete daughter molecules. A
simplified version of replication is shown in figure 9.5 and includes
the following:
1. uncoiling the parent DNA molecule, beginning at a predetermined point of origin;
2. unzipping the hydrogen bonds between the base pairs, thus
separating the two strands and exposing the nucleotide sequence of each strand (which is normally buried in the center
of the helix) to serve as templates; and
Parental helix
GC
AT
TAKE NOTE: THE SIGNIFICANCE OF DNA

STRUCTURE
1. Maintenance of the genetic codes during reproduction.

The constancy of base-pairing guarantees that the codes
will be retained during cell growth and division. When the
two strands are separated, each one provides a template
(pattern or model) for the replication (exact copying) of a
new molecule (gure 9.5). Because the sequence of one
strand automatically gives the sequence of its partner, the
codes can be duplicated with delity.
2. Providing variety. The order of bases along the length of
the DNA strand provides the information needed to produce RNA and protein molecules, which in turn are responsible for the phenotype of the cell. As we will see,
changing the identity of the bases or their order in the
DNA molecule can have a dramatic effect on the phenotype of the organism.
C
AT
AT
GC
G C
AT
AT
AT
AT
T A
CG
C
T A
AT
TA
CG
C
TA
AT
3
Replication fork
G
GC
Parental
New
GC
New
Replicas
5
Parental
Figure 9.5 Simplied steps to show the

semiconservative replication of DNA. The two strands of the
parental double helix are unwound and separated. Each single strand
will serve as a template (blue) to synthesize a new strand of DNA
(purple), starting at the replication fork. As synthesis proceeds, the
correct nucleotides are added according to the pattern of the template.
An A on the template will pair with a T on the new molecule, and a
C will pair with a G. The resultant new DNA molecules contain one
strand of the newly synthesized DNA and the original template
(parental) strand. This keeps the code intact because the same linear
arrangement of thebases is maintained during this process. Note that
the ne details of the process are presented in gure 9.6.
TABLE 9.1 Some Enzymes Involved in DNA

Replication and Their Functions
Enzyme
Function
Helicase
Primase
DNA polymerase III
Unzipping the DNA helix

Synthesizing an RNA primer
Adding bases to the new DNA chain;
proofreading the chain for mistakes
Removing RNA primer, closing gaps,
repairing mismatches
Final binding of nicks in DNA during
synthesis and repair
Supercoiling
DNA polymerase I
Ligase
Gyrase
3. synthesizing two new strands by attachment of the correct

complementary nucleotides to each single-stranded template.
A critical feature of DNA replication is that each daughter molecule
will be identical to the parent in composition, but neither one is
completely new; the strand that serves as a template is an original

parental DNA strand. The preservation of the parent molecule in
this way, termed semiconservative replication, helps explain the
reliability and fidelity of replication (figure 9.5).
table 9.1). The DNA polymerase III is responsible for deciphering

and duplicating DNA codes, but it has some major restrictions that
will affect the overall process (figure 9.6):

Events in Replication
All chromosomes have a specific origin of replication site that
serves as the place where replication will be initiated. It is recognized by a short sequence rich in adenine and thymine that, you will
recall, are held together by only two hydrogen bonds rather than
three. Because the origin of replication is AT-rich, less energy is
required to separate the two strands than would be required if the
origin were rich in guanine and cytosine.
The process of synthesizing a new daughter strand of DNA using the parental strand as a template is carried out by a giant enzyme complex that brings in the primary replication enzymeDNA
polymerase IIIalong with numerous accessory enzymes (see
4. Before synthesis of the lagging strand
can start, a primase first constructs a
short RNA primer to direct the DNA
polymerase III. Synthesis can proceed
only in short sections and produces
segments of RNA primer and new DNA
called Okazaki fragments.
DNA polymerase cannot add nucleotides to a DNA strand

without a preexisting unbound nucleotide.
Before replication can begin, a piece of RNA called a primer is
inserted at the origin of replication to serve as the starting point
for bases to be added.
The polymerase can add nucleotides only in the direction of 59
to 39, which means that synthesis of the two template strands
will be somewhat different.
Prior to the start of replication, enzymes called helicases (unzipping enzymes) bind to the DNA at the origin. These enzymes untwist the helix and break the hydrogen bonds holding the two
strands together, resulting in two separate strands, each of which
will be used as a template for the synthesis of a new strand (figure
9.6, step 1).
5. A second polymerase
(DNA polymerase I) acts
on the Okazaki fragments
by removing the primers.
i
zak fragm
en
t
3
6. Open spaces in the

lagging strand are filled in
by a ligase that adds the
correct nucleotides.
6
Oka
3. The template for the lagging strand

runs the opposite direction (3 to 5) and
must be replicated backwards away
from the replication fork so the DNA
polymerase can add the nucleotides in
the necessary 5 to 3 arrangement.
1
261
5
3
Lagging strand synthesis

3
3
5
5
4
1. The chromosome to be
replicated is continuously
unwound by a helicase,
forming a replication fork
with two template strands.
2. The template for the leading strand (bottom) is correctly

oriented for the DNA polymerase III to add nucleotides in
the 5 to 3 direction towards the replication fork, so it can
be synthesized as a continuous strand. Note that direction
of synthesis refers to the order of the new strand (red).
(b)
Leading strand synthesis

3
5
5
3
Key:
Replication forks
Template strand
Primase
New strand
DNA polymerase III
RNA primer
DNA polymerase I
Helicase
Ligase
(a)
Process Figure 9.6 The Assembly line of DNA replication in a circular bacterial chromosome. (a) A bacterial
chromosome showingthe overall pattern of replication. There are two replication forks where new DNA is being synthesized. (b) An enlarged
view of the leftreplication fork to show the details of replication.
262
Replication begins when an RNA primer is synthesized by a

primase at the origin of replication (figure 9.6 a). DNA polymerase
III must have this short strand of RNA to serve as a starting point for
adding nucleotides. Because the bacterial DNA molecule is circular,
opening of the circle forms two replication forks, each containing its
own set of replication enzymes. Each fork contains two active DNA
polymerases along with several other proteins and enzymes whose
main functions are to stabilize the polymerase and provide a means
of removing improperly incorporated nucleotides. The enzyme complex encircles the DNA near the replication fork and synthesizes new
DNA using the parent strand as a template. As synthesis proceeds,
the forks continually open up to expose the template for replication.
Because DNA polymerase III can only synthesize new DNA in
the 59 to 39 direction, just one strand, called the leading strand, can be
synthesized continuously (figure 9.6, step 2). One enzyme does this as
it moves along the template strand toward the replication fork. The
other strand, which is oriented 39 to 59 with respect to the polymerase,
is termed the lagging strand (figure 9.6, steps 3 and 4). Because this
strand cannot be synthesized continuously, an RNA primer is placed
ahead of the polymerase to initiate synthesis. It then adds nucleotides
a few at a time in the direction away from the fork (59 to 39). As the fork
opens up a bit, the next segment is synthesized backward near the point
of the previous segment. In this way, the two new strands can be
synthesized simultaneously. This manner of synthesis produces

short fragments of DNA (100 to 1,000 bases long) called Okazaki
fragments on the side of the lagging strand.
The lagging strand will be completed when the enzyme, DNA
polymerase I, removes the RNA primers from the Okazaki fragments and a ligase follows behind and inserts the correct DNA nucleotides into the open spaces (figure 9.6, steps 5 and 6). The final
result should be identical to the leading strand.
The addition of nucleotides proceeds at an astonishing pace, estimated in some bacteria to be 750 bases per second at each fork! As
replication proceeds, one newly synthesized strand loops down (figure
9.7a). When the forks have gone full circle, a termination site shuts
replication down. The two circular daughter molecules remain connected briefly but are nicked and separated by a helicase (figure 9.7b).
Like any language, DNA is occasionally misspelled when an
incorrect base is added to the growing chain. Studies have shown
that such mistakes are made once in approximately 108 to 109 bases,
but most of these are corrected. If not corrected, they are referred to
as mutations and can lead to serious cell dysfunction. Because continued cellular integrity is very dependent on accurate replication,
cells have evolved their own proofreading function for DNA. DNA
polymerase III, the enzyme that elongates the molecule, can also
detect incorrect, unmatching bases; excise them; and replace them
with the correct base. DNA polymerase I is involved in proofreading the molecule and repairing damaged DNA.
Forks
&
Check
Assess Section 9.1
Genetics is the study of the expression of biological information

(a)
Nick
Daughter cell
Daughter cell
(b)
Figure 9.7 Completion of chromosome replication in

bacteria. (a) As replication proceeds, one strand loops down.
(b) Final separation is achieved when an enzyme cuts and releases
the two completed molecules. The daughter cells receive these
during binary ssion.
and its transfer between organisms.

Nucleic acids are molecules that contain the blueprints of life in the
form of genes. DNA is the blueprint molecule for all cellular organisms. The blueprints of viruses, however, can be either DNA or RNA.
The total amount of DNA in an organisms chromosomes is termed
its genome. The genotype of each species contains a unique arrangement of genes that, when expressed, result in its traits or phenotype, including metabolic activities and pattern of reproduction.
The genome of prokaryotes is quite small compared with the genome of eukaryotes. A bacterial chromosome usually consists of a
few hundreds to thousands of genes. Eukaryotic genomes range
from thousands to tens of thousands of genes. Their DNA is packaged in tightly wound spirals arranged in discrete chromosomes.
DNA is a very long molecule composed of small subunits called
nucleotides. The sequence of the nucleotides contains information
needed to direct the synthesis of all proteins in the cell.
The DNA molecule must be copied so that the genetic material can
be transferred to offspring.
DNAs chemical structure makes its replication possible.
DNA copies itself just before cellular division by the process of
semiconservative replication. Semiconservative replication means
that each old DNA strand is the template upon which each new
strand is synthesized.
The circular bacterial chromosome is replicated at two forks as
directed by DNA polymerase III. At each fork, two new strands are
synthesizedone continuously and one in short fragments, and
mistakes are proofread and removed.
263
9.2 Applications of the DNA Code: Transcription and Translation
(a)
1. Compare the genetic material of eukaryotes, bacteria, and viruses

in terms of general structure, size, and mode of replication.
2. Characterize the organization of genetic material, going from the
genome down to the gene.
3. Briefly describe how DNA is packaged to fit inside a cell.
4. What are the fundamental building blocks of DNA and how are
they bonded together?
5. Describe what is meant by the antiparallel arrangement of DNA.
6. Explain the synthesis of the leading and lagging strands during
DNA replication and identify the enzymes required for producing
a finished product.
7. Name several characteristics of DNA structure that enable it to be
replicated with such great fidelity generation after generation.
DNA
Transcription of DNA
DSRNA
xpected Learning Outcomes
mRNA
rRNA
Translation of RNA
Ribosome
(rRNA + protein)
7. Present an overview of the main aspects of the ow of

genetic information in cells.
tRNA
8. Explain the relationship between the structure of DNA and

the structure of proteins.
mRNA
9. Describe the different types of RNA and their basic functions

in genetic expression.
10. Explain what happens during transcription.
11. Describe the genetic code, codons, and anticodons, and
how they relate to one another.
12. Relate the participants and steps in translation (protein
synthesis).
13. Distinguish major points of difference between prokaryotic
and eukaryotic transcription and translation.
We have explored how the genetic language of the DNA molecule

is conserved through replication. Next, we consider exactly how
DNA is expressed. Given that the sequence of bases in DNA is a
genetic code, just what is the nature of this code and how is it utilized? Although the genome is full of critical information, the
DNA molecule itself cannot perform cell processes directly. Instead, its information is conveyed to RNA molecules, which carry
out the instructions it contains. The concept that genetic information flows from DNA to RNA to protein has long been a central
theme of molecular biology (figure 9.8 a). It states that the master
code of DNA is first used to synthesize RNA via a process called
transcription, and the information contained in the RNA is then
used to produce proteins in a process known as translation. The
principal exceptions to this pattern are found in RNA viruses,
which convert RNA to other RNA, and in retroviruses, which convert RNA to DNA.
We now know that this so-called central dogma, though accurate, is somewhat incomplete. In addition to the RNA used to
produce proteins, a wide variety of specialized RNAs act by
Regulatory
RNAs
SSRNA
tRNA
9.2 Applications of the DNA Code:

Transcription and Translation
(b)
Protein
Micro RNA,
interfering RNA,
antisense RNA, and
riboswitches
regulate transcription
and translation
Expression of DNA
for structures and
functions of cell
Figure 9.8 Summary of the ow of genetic information

in cells. DNA is the ultimate storehouse and distributor of genetic
information. (a) DNA must be deciphered into a usable cell language.
It does this by transcribing its code into RNA helper molecules that
translate that code into protein. (b) Other sections of the DNA
produce additional types of RNA molecules that regulate genes and
their products.
regulating gene function (figure 9.8b). We will introduce some of

the expanded functions for RNA in Insight 9.4, but we will start
with its direct participation in protein synthesis.
The Gene-Protein Connection

The Triplet Code and the Relationship to Proteins
Several questions invariably arise concerning the relationship between genes and cell function. For instance, how does gene structure lead to the expression of traits in the individual, and what
features of gene expression cause one organism to be so distinctly
different from another? For answers, we must turn to the correlation
264
Triplets
1
Single nucleotide
3
4
DNA
mRNA
(copy of
one strand)
Codon
1
Amino acids
The Major Participants in Transcription

and Translation
Transcription, the formation of RNA using DNA as a template, and
translation, the synthesis of proteins using RNA as a template, are
highly complex. A number of components participate: most prominently, messenger RNA, transfer RNA, ribosomes, several types of
enzymes, and a storehouse of raw materials. After first examining
each of these components, we shall see how they come together in
the assembly line of the cell.
RNAs: Tools in the Cells Assembly Line

1
Variations in the order and types

will dictate the shape
and function of the protein.
Figure 9.9 Simplied view of the DNA-protein

relationship. The DNA molecule is a continuous chain of base pairs,
but the sequence must be interpreted in groups of three base pairs (a
triplet). Each triplet as copied into mRNA codons will translate into one
amino acid; consequently, the ratio of base pairs to amino acids is 3:1.
between gene and protein structure. We know that each structural

gene is an ordered sequence of nucleotides that codes for a protein.
Because each protein is different, each gene must also differ somehow in its composition. In fact, the language of DNA exists in the
order of groups of three consecutive bases, or triplets, on one DNA
strand (figure 9.9). In short, one gene differs from another in its
order of triplets. An equally important part of this concept is that
each triplet represents a code for a particular amino acid. When the
triplet code is transcribed and translated, it dictates the type and
order of amino acids in a polypeptide (protein) chain.
The final key points that connect DNA and protein function are:
1. DNA is a blueprint that indicates which kinds of proteins to
make and how to make them. This blueprint exists in the order
of triplets along the DNA strands.
2. The order of triplets directs a proteins primary structurethe
order and type of amino acids in the chainwhich determines
its characteristic shape and function.
3. Proteins contribute significantly to the phenotype by functioning as enzymes and structural molecules.
Ribonucleic acid (RNA) is an encoded molecule like DNA, but its

general structure is different in several ways:
1. It is a single-stranded molecule that can assume secondary and
tertiary levels of complexity due to bonds within the molecule,
leading to specialized forms of RNA (mRNA, tRNA, and
rRNA see figure 9.8).
2. RNA contains uracil, instead of thymine, as the complementary base-pairing mate for adenine. This does not change the
inherent DNA code in any way because the uracil still follows
the pairing rules.
3. Although RNA is a single helix that consists of alternating
sugar and phosphate molecules, the sugar in RNA is ribose
rather than deoxyribose (see figure 2.24).
The many functional types of RNA range from small regulatory
pieces to large structural ones. All types of RNA are formed through
transcription of a DNA gene, but only mRNA is further translated
into protein. See table 9.2 for a comparison of major types of RNA
and its functions. Other forms of RNA are introduced in section 9.3.
Messenger RNA: Carrying DNAs Message

Messenger RNA (mRNA) is a transcribed version of a structural
gene or genes in DNA. It is synthesized by a process similar to
synthesis of the leading strand during DNA replication, and the
complementary base-pairing rules ensure that the code will be
faithfully copied in the mRNA transcript. The message of this transcribed strand is later read as a series of triplets called codons
(figure 9.10a), and the length of the mRNA molecule varies from
about 100 nucleotides to several thousand. The details of transcription and the function of mRNA in translation are covered shortly.
TABLE 9.2 Major Types of Ribonucleic Acid Involved in Protein Synthesis

RNA Type
Contains Codes For
Function in Cell
Translated
Messenger (mRNA)
Sequence of amino acids in protein
Carries the DNA master code

to the ribosome
Yes
Transfer (tRNA)
A cloverleaf tRNA to carry amino acids
Brings amino acids to ribosome

during translation
No
Ribosomal (rRNA)
Several large structural rRNA molecules
Forms the major part of a ribosome

and participates in protein synthesis
No
Primer
An RNA that can begin DNA replication
Primes DNA
No
(a) Messenger RNA (mRNA)

A short piece of messenger
RNA (mRNA) illustrates the
general structure of RNA:
single strandedness,
repeating phosphate-ribose
sugar backbone attached to
single nitrogen bases; use
of uracil instead of thymine.
Codon 1
Codon 2
Codon 3
= Phosphate R = Ribose U = Uracil
Amino acid
attachment site
(b) Transfer RNA (tRNA)

Left: The tRNA strand loops back
on itself to form intrachain
hydrogen bonds. The result is a
cloverleaf structure, shown here
in simplified form. At its bottom is
an anticodon that specifies the
attachment of a particular amino
acid at the 3 end. Right: A
three-dimensional view of tRNA
structure.
5
G
C
G
G
A
U
U
U
A
H bonds
G AC
G
G
G A
G AG
C
C
C
A
G
CA
U
U A
Figure 9.10
265
Amino acid
attachment site
A
C
G
C
U
U
A
C U
A
GA C A C
UGU G
C
T
U
G
A
G
5
3
G
C
Hairpin
loops
G
U
C
A
C
Anticodon
Anticodon
Characteristics of messenger and transfer RNA.
Transfer RNA: The Key to Translation

Transfer RNAs (tRNA) are also complementary copies of specific
regions of DNA; however, they differ from mRNA. TRNA is uniform in length, being 75 to 95 nucleotides long, and it contains sequences of bases that form hydrogen bonds with complementary
sections of the same tRNA strand. At these points, the molecule
bends back upon itself into several hairpin loops, giving the molecule a secondary cloverleaf structure that folds even further into a
complex, three-dimensional helix (figure 9.10b). This compact
molecule acts as a translator that converts RNA language into protein language. The bottom loop of the cloverleaf exposes a triplet,
the anticodon, that both designates the specificity of the tRNA and
complements mRNAs codons. At the opposite end of the molecule
is a binding site for the amino acid that is specific for that tRNAs
anticodon. For each of the 20 amino acids, there is at least one specialized type of tRNA to carry it. Binding of an amino acid to its
specific tRNA requires a specific enzyme that can correctly match
each tRNA with its amino acid.
The Ribosome: A Mobile Molecular Factory

for Translation
The prokaryotic (70S) ribosome is a particle composed of tightly
packaged ribosomal RNA (rRNA) and protein. The rRNA component of the ribosome is also a long polynucleotide molecule. It forms
complex three-dimensional figures that contribute to the structure and
function of ribosomes. The interactions of proteins and rRNA create
the two subunits of the ribosome that engage in final translation of
thegenetic code (see figure 9.12). A metabolically active bacterial cell

can accommodate up to 20,000 of these minuscule factoriesall
actively engaged in reading the genetic program, taking in raw materials, and producing proteins at an impressive rate.
Transcription: The First Stage

of Gene Expression
Like so many other genetic events, transcription is a marvel of efficiency. It is highly organized and guided by special codes on DNA
itself. To describe it in more detail, we use the formation of mRNA
in bacteria as a model. It is important to keep in mind that all RNA
(tRNA, rRNA) is synthesized by a similar process. The other RNAs
have important functions; they just do not provide the code for the
amino acids in a protein as mRNA does.
During transcription, an RNA molecule is synthesized using
the codes on DNA as a guide or template. A large enzyme complex,
RNA polymerase, is responsible for this process. This polymerase
is more multipurpose than DNA polymerase, because the RNA enzyme works alone and does not require a helicase. It can bind to
DNA and unwind it, as well as synthesize RNA (figure 9.11).
Transcription proceeds in three stages: initiation, elongation, and
termination. Initiation requires the RNA polymerase to recognize a
region on a gene called the promoter region (figure 9.11, step 1). This
region consists of two sets of DNA sequences located just before the
initiation site. The first sequence is about 35 nucleotides from the initiation site, and the second is about 10 nucleotides from it. The primary
function of the promoter is to provide a position for initial binding of
1 Each gene contains a

specific promoter region
and a leader sequence for
guiding the beginning of
transcription. Next is the
region of the gene that
codes for a polypeptide
and ends with a series of
terminal sequences that
stop translation.
RNA polymerase binding site

Initiation
Leader
sequence codon
Termination
sequences
Promoter
region
T A C
A T G
RNA
polymerase
G A
C T
C
G
2 DNA is unwound at
the promoter by RNA
polymerase. Only one
strand of DNA, called
the template strand,
is copied by the RNA
polymerase. This
strand runs in the 3
to 5 direction.
G A T
C T A
G C
C G(
Intervening sequence of variable size

Termination sequence
Template strand
3
T
A
266
3
Nontemplate strand
Unwinding of DNA
Direction of
transcription
3 The RNA polymerase

moves along the
strand, adding
complementary
nucleotides as
dictated by the DNA
template. The mRNA
strand reads in the 5
to 3 direction.
Nucleotide
pool
Early mRNA
transcript
Elongation
4 The polymerase
continues transcribing
until it reaches a
termination site, and
the mRNA transcript
is released to be
translated. Note that
the section of the
transcribed DNA is
rewound into its
original configuration.
Process Figure 9.11
Late mRNA transcript
The major events in transcription, using mRNA as a model.
the RNA polymerase. The promoter sequences are not highly varied,
and they tend to be rich in adenine and thymine base pairs, which
have one less hydrogen bond, thus facilitating separation of the
DNA strands.
Prior to the first synthesis step of transcription, the RNA polymerase begins to separate the two strands of the DNA helix and
forms an open bubble of sorts (figure 9.11, steps 2 and 3). This
bubble serves as the space where the bonds between the nucleotides
of mRNA will actually be made. Only one strand of DNAthe
template strandis transcribed. This is the one that carries a message that can be translated into a protein. The other strandthe
nontemplate strandcan serve a genetic function but not as a
message for protein structure. Which strand becomes the template
varies from one gene to another. Note that the promotor sequences
are not transcribed as part of the final mRNA molecule.
The first DNA triplet to be transcribed is almost invariably
TAC. Recall that, since RNA will have uracil, not thymine, as the
complement of adenine, this means that the first codon on the
mRNA will be AUG (see start codon in figure 9.13). As elongation

proceeds, the polymerase moves the transcription bubble forward,
exposing subsequent sections of DNA. It simultaneously brings in
nucleotides that are complementary to the DNA template and continues to assemble the mRNA strand. As with replication, this occurs in the 59 to 39 direction with regard to the growing mRNA.
DNA that has already been transcribed rewinds back into its double
helix structure (figure 9.11, step 4). The part of the mRNA strand
that is already assembled remains attached to the enzyme complex
but hangs out of the way of the processing machinery.
At termination, the polymerase recognizes another code on DNA
near the end of the gene that signals the separation and release of the
completed mRNA. It will go immediately to the translation phase on
the ribosome. As we will see, this mRNA delivers its message in
codons that are the master code for protein synthesis. The length of
the final mRNA molecule depends upon the polypeptide that it
encodes. The smallest one may consist of 100 nucleotides, an average one of 1,200 nucleotides, and the largest several thousand.
267
Large
subunit
Amino acids
Exit site
P
E
Small
subunit
5
tRNAs
mRNA
transcript
Figure 9.12 The players in translation. A ribosome serves

as the stage for protein synthesis. Assembly of the small and large
subunits results in specic sites for holding the mRNA and two tRNAs
with their amino acids.
Initiation of Translation
The mRNA molecule leaves the DNA transcription site and is transported directly to ribosomes. Ribosomal subunits are specifically assembled in a way that forms sites to hold the mRNA and tRNAs. The
ribosome thus recognizes these molecules and stabilizes reactions between them. The small subunit binds to the 59 end of the mRNA, and the
large subunit supplies enzymes for making peptide bonds on the protein.
The ribosome begins to scan the mRNA by moving in the 59 to 39 direction along the mRNA. Translation begins when the ribosome encounters thestart codon, which is almost always AUG (and rarely, GUG).
With the mRNA message in place on the assembled ribosome,
the next step in translation involves entrance of tRNAs with their
amino acids. The pool of cytoplasm around the region contains a
complete array of tRNAs, previously charged by having the correct
amino acid attached. The step in which the complementary tRNA
meets with the mRNA code is guided by the two sites on the large
subunit of the ribosome called the P site (left) and the A site (right).1
Think of these sites as recessed spaces tucked within the two subunits of the ribosome, with each site accommodating a tRNA. The
ribosome also has an exit or E site where used tRNAs are released.
Translation: The Second Stage

of Gene Expression
The Master Genetic Code: The Message

in Messenger RNA
In translation, all of the elements needed to synthesize a protein, from

the mRNA to the tRNAs with amino acids, are brought together on
the ribosomes (figure 9.12). The process occurs in five stages: initiation, elongation, termination, and protein folding and processing.
By convention, the master genetic code is represented by the

mRNA codons and the amino acids they specify (figure 9.13).
1. P stands for peptide site; A stands for aminoacyl (amino acid) site; E stands for exit site.
Second Base Position
UUU
UUC
U
UUA
First Base Position
UUG
}
}
C
Phenylalanine
Leucine
UCU
UAU
UCC
UAC
Serine
UCA
UAA
UCG
UAG
CUU
CCU
CAU
CUC
CCC
CAC
Proline
Leucine
C
CUA
CCA
CAA
CUG
CCG
CAG
AUU
Isoleucine
AUC
ACU
AAU
ACC
AAC
Threonine
A
AUA
ACA
AAA
AUG* START fMethionine
ACG
AAG
GUU
GCU
GAU
GUC
GCC
GAC
Valine
Alanine
GUA
GCA
GAA
GUG
GCG
GAG
}
}
}
}
}
}
}
}
Tyrosine
G
UGU
UGC
STOP**
Histidine
Cysteine
U
C
UGA
STOP**
UGG
Tryptophan
CGU
CGC
C
Arginine
Glutamine
Asparagine
CGA
CGG
AGU
AGC
AGA
Lysine
Aspartic acid
AGG
}
}
Serine
U
C
Arginine
Third Base Position
A
G
GGU
GGC
C
Glycine
Glutamic acid
GGA
GGG
* This codon initiates translation.

**For these codons, which give the orders to stop translation, there are no corresponding tRNAs with amino acids.
Figure 9.13 The genetic code: codons of mRNA that specify a given amino acid. The master code for translation is provided by
mRNA codons.
268
DNA
triplets
mRNA
codons
G
A T
A C
T
G
A U
tRNA
UAC
anticodons
C
G
C T
A
G A
T
Nontemplate
strand
A C G
T G C
C U
Template strand
GA C
enters the P site and binds to the start codon (AUG) presented by
the mRNA. Rules of pairing dictate that the anticodon of this tRNA
will complement the mRNA codon AUG; thus, the tRNA with anticodon UAC will first occupy site P. It happens that the amino acid
carried by the initiator tRNA in bacteria is formyl methionine (fMet;
see figure 9.13), though in many cases, it may not remain a permanent part of the finished protein.
A C G
UGA
UGC
Threonine
Threonine
Leucine
F-Methionine
Protein
(amino
acids
specified)
Same amino acid; has a

different codon and anticodon
Figure 9.14 Interpreting the DNA code. If the DNA

sequence is known, the mRNA codon can be surmised. If a codon is
known, the anticodon and, nally, the amino acid sequence can be
determined. The reverse is not possible (determining the exact codon
or anticodon from amino acid sequence) due to the redundancy of
the code.
Continuation and Completion of Protein

Synthesis: Elongation and Termination
The mRNA formed during transcription provides a specific order of
nucleotides (codons) called the reading frame that must be translated in that exact order. In general, the ribosome shifts its reading
frame along the mRNA from one codon to the next. This brings
each succeeding codon into place on the ribosome and makes a
space for the next tRNA to enter. Then, a peptide bond is formed
between the amino acids on the adjacent tRNAs, and the polypeptide grows in length. The process is outlined here:
Except in a very few cases (the genes of mitochondria and chloroplasts, for example), this code is universal, whether for prokaryotes, eukaryotes, or viruses. It is worth noting that once the triplet
code on mRNA is known, the original DNA sequence, the complementary tRNA code, and the types of amino acids in the protein
are automatically known (figure 9.14). However, one cannot predict (backward) from protein structure what the exact mRNA codons are because of a factor called redundancy or degeneracy,
meaning that a particular amino acid can be coded by more than
one codon.
In figure 9.13, the mRNA codons and their corresponding
amino acid specificities are given. Because there are 64 different
triplet codes2 and only 20 different amino acids, it is not surprising
that some amino acids are represented by several codons. For example, leucine and serine can each be represented by any of six
different triplets, and only tryptophan and methionine are represented by a single codon. In such codons as leucine, only the first
two nucleotides are required to encode the correct amino acid, and
the third nucleotide does not change its sense. This property, called
wobble, is thought to permit some variation or mutation without
changing the message.
The Beginning of Protein Synthesis

With mRNA serving as the guide, the stage is finally set for actual
protein assembly. The correct tRNA (labeled 1 on figure 9.15)
2. 64 5 43 (the 4 different nucleotides in all possible combinations of 3).
Elongation begins with the filling of the A site by a second

tRNA (figure 9.15, step 1). The identity of this tRNA and its
amino acid is dictated by the second mRNA codon.
The entry of tRNA 2 into the A site brings the two adjacent
tRNAs in favorable proximity for a peptide bond to form between the amino acids (aa) they carry. The fMet is transferred
from the first tRNA to aa 2, resulting in two coupled amino
acids called a dipeptide (figure 9.15, step 2).
For the next step to proceed, some room must be made on the
ribosome, and the next codon in sequence must be brought into
position for reading. This process is accomplished by translocation, the enzyme-directed shifting of the ribosome to the
next position on the mRNA strand, which causes the blank
tRNA (1) to be discharged from the ribosome (figure 9.15,
step 3) at the E site.
This shifts the tRNA holding the dipeptide into P position. Site
A is temporarily left empty. The tRNA that has been released
is now free to drift off into the cytoplasm and become recharged with an amino acid for later additions to this or another
protein.
The stage is now set for the insertion of tRNA 3 at site A as directed by the third mRNA codon (figure 9.15, step 4). This insertion is followed once again by peptide bond formation
between the dipeptide and aa 3 (making a tripeptide), splitting of
the peptide from tRNA 2, and translocation (figure 9.15, step 5).
This releases tRNA 2, shifts mRNA to the next position, moves
tRNA 3 to position P, and opens position A for tRNA 4
(step 6). From this point on, peptide elongation proceeds repetitively by this same series of actions out to the end of the
mRNA (steps 7 and 8).
The termination of protein synthesis is not simply a matter of

reaching the last codon on mRNA. It is brought about by the presence of at least one special codon occurring just after the codon for
the last amino acid. Termination codonsUAA, UAG, and UGA
are codons for which there is no corresponding tRNA. Also termed
nonsense or stop codons, they carry a necessary message: Stop
here. When this codon is reached, a special enzyme breaks the bond
Leucine
f Met
Peptide bond 2
3
2
1
A
P
E
Anticodon
G C
A
C UG
GCU
C CG
A UG
mRNA
AU
AUC
U AC
G C
A
C UG
G C
G
C CG
G CU
A
UC
Start
codon
1 Entrance of tRNAs 1 and 2
AG
Peptide bond 1
UAG
5 Formation of peptide bond
Alanine
1
A
P
E
2
1
1
G C
A
C UG
C
GA
C
A
P
GA
E
UAG
G
GC
CC G
Empty tRNA
GC U
AU C
AUG C U G
AUC
U AC
A UG
GCU
C CG
UAG
6 Discharge of tRNA 2; second

translocation; tRNA 4 enters ribosome
P
UAC
G AC
C UG
C CG
GCU
AUC
A UG
Peptide bond 3
4
3
A
P site
3 Discharge of tRNA 1 at E site
UAG
3
G C
G
CC G
Proline
C A
G
GC U
AUC
AUG C U G
UA G
UAG
G C
G
E
Stop codon
AU
G C
A
C UG
C CG
GC
Steps 3 to 5 are repeated until stop codon

is reached and protein synthesis is
terminated.
4 First translocation; tRNA 2 shifts into

P site; tRNA 3 enters ribosome at A
UC
U A
A site
UAG
Process Figure 9.15
The events in protein synthesis.
269
270
between the final tRNA and the finished polypeptide chain, releasing it from the ribosome.
Before newly made proteins can carry out their structural or
enzymatic roles, they often require finishing touches. Even before the peptide chain is released from the ribosome, it begins
folding upon itself to achieve its biologically active tertiary conformation. Other alterations, called posttranslational modifications, may be necessary. Some proteins must have the starting
amino acid (formyl methionine) clipped off; proteins destined
to become complex enzymes have cofactors added; and some
join with other completed proteins to form quaternary levels of
structure.
The operation of transcription and translation is machinelike in its precision. Protein synthesis in bacteria is both efficient
and rapid. At 37C, 12 to 17 amino acids per second are added
to a growing peptide chain. An average protein consisting of
about 400amino acids requires less than half a minute for complete synthesis. Further efficiency is gained when the translation
of mRNA starts while transcription is still occurring (figure
9.16). A single mRNA is long enough to be fed through more
than one ribosome simultaneously. This permits the synthesis of
hundreds of protein molecules from the same mRNA transcript
arrayed along a chain of ribosomes. This polyribosomal complex is indeed an assembly line for mass production of proteins.
Protein synthesis consumes an enormous amount of energy.
Nearly 1,200 ATPs are required just for synthesis of an averagesize protein.
RNA polymerase
mRNA
Transcription
Start of
translation
(a)
Growing
polypeptides
1
2
Polyribosomal
complex
3
4
5
Start
(b)
Eukaryotic Transcription and Translation:

Similar yet Different
Eukaryotes share many aspects of protein synthesis with
prokaryotes. They show similarities in the operation of the ribosomes, the functions of start and stop codons, and the mass
production of proteins by means of polyribosomes. But they do
differ in significant ways. The start codon in eukaryotes is also
AUG, but it codes for an alternate form of methionine. Another
difference is that eukaryotic mRNAs code for just one protein,
unlike bacterial mRNAs, which often contain information from
several genes in series.
Prokaryotic and eukaryotic cells also vary in location and
structure of genes. The presence of DNA in the nucleus of eukaryotic cells means that eukaryotic transcription and translation cannot
be simultaneous as it is in prokaryotes. The mRNA transcript must
pass through pores in the nuclear membrane and be carried to the
ribosomes in the cytoplasm or on the endoplasmic reticulum for
translation.
We have given the simplified definition of a gene that works
well for most prokaryotes,3 but eukaryotic genes are generally not
colinear4meaning that they do not exist as an uninterrupted series
of triplets coding for a protein. A eukaryotic gene contains the code
for a protein, but located along the gene are one to several intervening
sequences of bases, called introns, that do not code for protein.
Introns are interspersed between coding regions, called exons, that
will be translated into protein (figure 9.17). We can use words as
examples. A short section of colinear prokaryotic gene might read
mRNA
mRNA
Ribosomes
Ribosomes
Polypeptide
Polypeptide
(c)
Figure 9.16 Speeding up the protein assembly line in

bacteria. (a) The mRNA transcript encounters ribosomal parts
immediately as it leaves the DNA. (b) The ribosomal factories
assemble along the mRNA in a chain, each ribosome reading the
message and translating it into protein. Many products will thus
be well along the synthetic pathway before transcription has even
terminated. (c) Photomicrograph of a polyribosomal complex in
action. Note that the protein tails vary in length depending on
the stage of translation (30,0003).
3. Introns have been found in cyanobacteria and archaea.

4. Colinearity means that the base sequence can be read directly into a series of
amino acids.
INSIGHT 9.3
Revising Some Rules of Genetics
What was true for E. coli would be true for the elephant.
Jacques Monod, Nobel laureate and discover of the lac operon
increasing or decreasing the expression of certain genes by making them

more or less accessible for transcription. For example, having methyl
groups (CH3) attached to numerous cytosine nucleotides reduces the
level of transcription of a section of DNA that is methylated.
A recent study in mice determined that the mothers diet during
gestation could greatly alter gene expression in her offspring. Genetically
identical mothers (clones) were placed into two groupsone that was fed
a diet high in the soy chemical genistein and the other a normal diet. The
soy group had children with significant differences in color coat and
weight. They were larger and yellower than the mice from the normal
group. This was a permanent effect, since the soy group remained unchanged even when placed on a normal diet. The effect appears to be due
to methylation of the embryonic genes by genistein during fetal development (see figure).
Bacteriawith their small genomes and short generation timeswere

the ideal model for research into molecular genetics. From this research
came elegant and universal rules governing the workings of DNA. This
led to the Central Dogma of Biology, which says that DNA is the repository of genetic information, RNA is a molecule that ferried information
between DNA and the ribosome, and proteins produced from the RNA
codes are responsible for carrying out the structural and metabolic needs
of the cell. And all of this is still accurate.
It is just that there is more to the story. Recent discoveries in eukaryotic organisms have revealed that certain aspects of their genetics have
turned out to be somewhat more complex and varied. This does not
change our introduction to microbial genetics for two reasons. First, the
laws of genetics covered in this chapter do hold true for the vast majority
of prokaryotic organisms. Second, this is an introduction to genetics and
it is certainly fair to leave the exceptions, the unusual, and the overly
complex for future courses. We would like to use this Insight to introduce
some of these revolutionary new findings.
90,000 Proteins . . . 25,000 Genes?

Humans have about 90,000 different proteins, and, going by our earlier
understanding, we would have expected at least 90,000 different genes.
Imagine the surprise most biologists felt when the Human Genome Project revealed that we have fewer than 25,000 protein-coding sequences,
about 15,000 fewer than a simple corn plant.
For some time, it has been known that eukaryotic genes consist of
expressed sequences (exons) interspersed with intervening sequences
(introns) and that the final mRNA need not contain every exon. Until our
stunningly low gene count was revealed, however, no one realized how
often a single gene could be used to make several different proteins. Utilizing alternative splicing, a cell can select which exons will be retained
and spliced into a final mRNA. Like a Scrabble player deciding which
lettered tiles to use to make a word, a cell can place any combination of
the exons in a gene to produce many alternate mRNAs (and resulting
proteins). It is currently estimated that three of every four human genes
are edited in this way and that the prevalence of alternative splicing appears to increase with the complexity of an organism.
The Genetic Code Is Universal with

Some Exceptions
Based on early studies, it was assumed that the genetic code was the same
in all cells and viruses, as no exceptions had ever been seen. Even more
convincing, eukaryotic mRNAs were appropriately translated by bacterial cells, arguing for a single genetic dictionary. But later work with
mitochondria isolated from yeast and humans found a slightly different
code; for example, the stop codon UGA specifies the amino acid tryptophan in mitochondria. This discovery was followed by the detection of
other exceptions to the genetic code in a few prokaryotic and eukaryotic
organisms. This difference in the genetic code of mitochondria is one of
the most compelling pieces of evidence supporting the endosymbiotic
theory (see Insight 5.1).
Can you give some reasons for such a large proportion of a genome
being apparently silenced or unused? Answer available at http://
www.mhhe.com/talaro8
Genetic Expression Can Be Modied

by Epigenetic Mechanisms
For years, geneticists have debated the
effects of nature (genes) versus nurture (environment) on the development of organisms. Though both have
profound influence, we now have
greater understanding about the direct
effects of nongenetic factors.
The term epigenetics is used to
designate factors that effect genetic
expression other than the specific sequence of nucleotides in genes. One
major epigenetic effect is in the manner that DNA coils around proteins
during chromatin formation (see Insight 9.1). This layer of information is
only now being fully appreciated and
is still far from being understood. Epigenetic effects seem to work by
NH2
NH2
H
N
O
Normal cytosine
CH3
N
O
Methylated cytosine
Section of gene with methylation that

blocks its activation
One example of epigenetic effects due to

methylation of bases during early development.
Mice from
f
mothers
h
ffed
dah
high-soy
h
d
diet were llarger
and different-colored from mice whose mothers
were fed a normal diet.
271
272
TOM SAW OUR DOG DIG OUT; a eukaryotic

E
E
E
E
I
I
I
DNA
gene that codes for the same portion would read
template
TOM SAW XZKP FPL OUR DOG QZWVP DIG
Exon Intron
OUT. The recognizable words are the exons, and
the nonsense letters represent the introns.
Primary
This unusual genetic architecture, also called a
E
E
E
E
I
I
I
mRNA
transcript
split gene, requires further processing before translation. Transcription of the entire gene with both
exons and introns occurs first, producing a preLariat forming Spliceosomes
mRNA. Next, a type of RNA and protein called a
Occurs
spliceosome recognizes the exon-intron junctions
in
nucleus
and enzymatically cuts through them in a process
Transcript
processed
called RNA splicing. The action of this splicer enE
E
E
E
by special
zyme loops the introns into lariat-shaped pieces,
enzymes
excises them, and joins the exons end to end. By
this means, a strand of mRNA with no intron mateLariat excised
rial is produced. This completed mRNA strand can
then proceed to the cytoplasm to be translated.
Exons
Spliceosomes released
Several different types of introns have been
spliced
discovered, some of which do code for cell subtogether
stances. A great deal of non-protein-coding DNA is
E
E
E
E
proving to be vital for cell function. In humans, this
intron material represents 98% of the DNA found
Occurs
in chromosomes. Another surprising discovery is
in
mRNA transcript can
cytoplasm
that some proteins have sections that are in reverse
now be translated
order from the DNA sequence that encoded them.
Previously, it was considered a hard and fast rule
Figure 9.17 The split gene of eukaryotes. Eukaryotic genes have an
that DNA sequence determined the mRNA and
additional complicating factor in their translation. Their coding sequences, or exons
consequently amino acid sequence after the introns
(E), are interrupted at intervals by segments called introns (I) that are not part of that
were removed. This new data revealed that cellular
proteins code. Introns are transcribed but not translated, which necessitates their
machinery can reverse the order of DNA, essen- removal by RNA splicing enzymes before translation.
tially creating new proteins from the same gene.
Many introns have been found to code for an enzyme called reverse
transcriptase, which can convert RNA into DNA. Other introns are
translated into endonucleases, enzymes that can snip DNA and allow insertions and deletions into the sequence. See Insight 9.3 for
Ribosomes are sites of protein assembly by mRNA and tRNA.
other examples of exceptional eukaryotic genetics.
The processes of transcription and translation are similar but not
identical for prokaryotes and eukaryotes.
&
Check
Assess Section 9.2
Information in DNA is converted to proteins by the process of
transcription and translation. Structural proteins contribute to the

architecture of the cell, while functional proteins (enzymes) control an organisms metabolic activities.
The codes on DNA are transferred to various RNA molecules,
which carry out numerous functions, including protein synthesis
and genetic regulation.
The DNA code occurs in groups of three bases called codons;
codes are transferred to RNA through transcription.
Messenger (m) RNA carries the master message that instructs
which amino acids are added during translation.
Transfer (t) RNA carries the amino acid that will match the correct
codon on mRNA.
8. How is the language of the gene expressed?

9. If a bacterial protein is 3,300 amino acids long, how many nucleotide pairs long is the gene sequence that codes for it?
10. Construct a table that compares the structure and functions of
DNA and RNA.
11. Where does transcription begin?
12. What are the template and nontemplate strands of DNA?
13. Why is only one strand transcribed, and is the same strand of DNA
always transcribed?
14. Distinguish between a codon and an anticodon.
15. Briefly describe the events in translation.
16. What are the functions of start and stop codons? Give examples.
17. Summarize how bacterial and eukaryotic cells differ in gene structure, transcription, and translation.
18. Discuss the roles of exons and introns.

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taL75292_ch09_254-290.

indd Page 254 11/12/10 11:16 PM user-f494

At War with an Emerging Pathogen

55-year-old woman being treated

multiple locations and cultured them. The

What is the probable origin of the

What does drug resistance mean from

To continue the case, go to page 285.

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9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity

9.1 Introduction to Genetics and Genes:

xpected Learning Outcomes

1. Dene heredity, genetics, genome, gene, phenotype, and

2. Discuss the basic nature of genetic material in eukaryotes,

Genetics is the science that studies the inheritance of biological

the transmission of biological properties (traits) from parent to

The study of genetics takes place on several levels (figure 9.1).

The Nature of the Genetic Material

The Levels of Structure and

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Chapter 9 Microbial Genetics

All chromosomes contain a series of basic informational

The Structure of DNA: A Double Helix

The Size and Packaging of Genomes

* genotype (jee9-noh-typ) Gr. gennan, to produce, and typos, type.

Figure 9.3 A disrupted Escherichia colicell has spewed out its

9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity

The condensation of DNA and protein into chromatin was once

Chemical tags attached to histone

DNA wrapped around

The packaging of DNA. Eukaryotic DNA is intimately

Chapter 9 Microbial Genetics

X rays produce a photographic image that can

bonds with guanine. This difference will influence a number of

taL75292_ch09_254-290.indd Page 259

One of the significant findings that has emerged from sequencing

DNA Replication: Preserving the Code

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Chapter 9 Microbial Genetics

The arrangement of nitrogen bases in DNA has two essential

It is tempting to consider how such a seemingly simple

The Overall Replication Process

TAKE NOTE: THE SIGNIFICANCE OF DNA

1. Maintenance of the genetic codes during reproduction.

Figure 9.5 Simplied steps to show the

TABLE 9.1 Some Enzymes Involved in DNA

Unzipping the DNA helix

3. synthesizing two new strands by attachment of the correct

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9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity

completely new; the strand that serves as a template is an original

table 9.1). The DNA polymerase III is responsible for deciphering

DNA polymerase cannot add nucleotides to a DNA strand

6. Open spaces in the

3. The template for the lagging strand

Lagging strand synthesis

2. The template for the leading strand (bottom) is correctly

Leading strand synthesis

DNA polymerase III

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Chapter 9 Microbial Genetics

Replication begins when an RNA primer is synthesized by a

synthesized simultaneously. This manner of synthesis produces