Microbial Genetics PDF

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CHAPTER
9
Microbial Genetics
V
CASE FILE ancomycin-resistant Staphylococcus aureus (VRSA) was isolated from the exit site of a
dialysis catheter in a 40-year-old diabetic with a history of peripheral vascular disease,
9 chronic renal failure, and chronic foot ulcers. A few months earlier, the patient’s gangre-
nous toe had been amputated. Following that surgery, the patient developed bacteremia with
methicillin-resistant S. aureus from an infected hemodialysis graft. Vancomycin, rifampin, and
graft removal successfully treated the infection.
A few months later, when the catheter exit site infection appeared, the area was cultured
and the catheter removed, successfully treating the infection. A week later, the patient’s chronic
foot ulcer again appeared infected. Vancomycin-resistant Enterococcus faecalis (VRE) and Klebsi-
ella oxytoca were cultured from the ulcer. The patient recovered after wound care and systemic
treatment with trimethoprim/sulfamethoxazole.
Analysis of the VRSA isolate revealed that it contained the vanA gene for vancomycin resis-
tance and the mecA gene for oxacillin resistance.
How do you think the Staphylococcus aureus strain ended up with the gene for vancomycin
resistance?
What is one possible mechanism for genetic transfer of antibiotic resistance from one organism
to another?
Why would this particular patient be at increased risk for infection with VRSA?
Case File 9 Wrap-Up appears on page 249.
CHAPTER OVERVIEW Genetics is the study of the transfer of information between biological entities. The
molecules most important to this endeavor are DNA, RNA (both of which carry
information), and proteins, which carry out most cellular functions and are built using
the information in DNA and RNA.
DNA is a very long molecule composed of small subunits called nucleotides. The
sequence of the nucleotides contains the information needed to eventually direct the
synthesis of all proteins in the cell.
Viruses contain various forms of DNA and RNA that are translated by the genetic
machinery of their host cells to form functioning viral particles.
The genetic activities of cells are highly regulated by operons, groups of genes that
interact as a unit to control the use or synthesis of metabolic substances, as well as by
RNA regulatory molecules.
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9.1 Introduction to Genetics and Genes: Unlocking the Secrets of Heredity 247
The DNA molecule must be replicated for the distribution of genetic material to
offspring. When replication is not faithful, permanent changes in the sequence of
the DNA, called mutations, can occur. Because mutations may alter the function or
expression of genes, they serve as a force in the evolution of organisms.
Bacteria undergo genetic recombination through the transfer of small pieces of DNA
between bacteria as well as the uptake of DNA from the environment.
9.1 Introduction to Genetics and Genes: The Nature of the Genetic Material
Unlocking the Secrets of Heredity For a species to survive, it must have the capacity of self-
replication. In single-celled microorganisms, reproduction
Genetics is the study of the inheritance, or heredity, of living involves the division of the cell by means of binary fission
things. It is a wide-ranging science that explores or budding, but these forms of reproduction involve a
more significant activity than just simple cleavage of the
1. the transmission of biological properties (traits) from cell mass. Because the genetic material is responsible for
parent to offspring; inheritance, it must be accurately duplicated and sepa-
2. the expression and variation of those traits; rated into each daughter cell to ensure normal function.
3. the structure and function of the genetic material; and This genetic material itself is a long molecule of DNA
4. how this material changes. that can be studied on several levels. Before we look at
The study of genetics takes place on several levels (figure 9.1). how DNA is copied, let us explore the organization of
Organismal genetics observes the heredity of the whole organ- this genetic material, proceeding from the general to the
ism or cell; chromosomal genetics examines the characteristics specific.
and actions of chromosomes; and molecular genetics deals
with the biochemistry of the genes. All of these levels are useful
areas of exploration, but in order to understand the expressions
The Levels of Structure and
of microbial structure, physiology, mutations, and pathogenic- Function of the Genome
ity, we need to examine the operation of genes at the cellular The genome is the sum total of genetic material of a cell.
and molecular levels. The study of microbial genetics provides Although most of the genome exists in the form of chromosomes,
a greater understanding of human genetics and an increased genetic material can appear in nonchromosomal sites as well
appreciation for the astounding advances in genetic engineer- (figure 9.2). For example, bacteria and some fungi contain
ing we are currently witnessing (see chapter 10). tiny extra pieces of DNA (plasmids), and certain organelles of
Organism level Cell level Chromosome level Molecular level
TA
GC
AT
C
Eukaryotes CG
TA
GC
A
TA
GC
CG
A
TA
CG
A
T
Prokaryotes 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).
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248 Chapter 9 Microbial Genetics
Cells Prokaryote
Eukaryote
(composite)
Chromosomes
Nucleus
Chromosome Plasmids
Mitochondrion
Plasmid
(in some
fungi and
Viruses
protozoa)
Extrachromosomal
DNA
Chloroplast DNA RNA
Figure 9.2 The general location and forms of the genome in selected cell types and viruses (not to scale).
eukaryotes (the mitochondria and chloroplasts) are equipped that control gene expression. The sum of all of these types of
with their own genetic programs. Genomes of cells are com- genes constitutes an organism’s distinctive genetic makeup,
posed exclusively of DNA, but viruses contain either DNA or or genotype (jee⬘-noh-tıp). The expression of the genotype
RNA as the principal genetic material. Although the specific creates traits (certain structures or functions) referred to
genome of an individual organism is unique, the general pat- as the phenotype (fee⬘-noh-t ıp). Just as a person inherits a
tern of nucleic acid structure and function is similar among all combination of genes (genotype) that gives a certain eye
organisms. color or height (phenotype), a bacterium inherits genes
In general, a chromosome is a discrete cellular struc- that direct the formation of a flagellum, and a virus con-
ture composed of a neatly packaged DNA molecule. The tains genes for its capsid structure. All organisms contain
chromosomes of eukaryotes and bacterial cells differ in more genes in their genotypes than are manifested as a
several respects. The structure of eukaryotic chromosomes phenotype at any given time. In other words, the pheno-
consists of a DNA molecule tightly wound around histone type can change depending on which genes are “turned
proteins, whereas a bacterial chromosome is condensed on” (expressed).
and secured into a packet by means of histonelike proteins.
Eukaryotic chromosomes are located in the nucleus; they
vary in number from a few to hundreds; they can occur The Size and Packaging of Genomes
in pairs (diploid) or singles (haploid); and they appear Genomes vary greatly in size. The smallest viruses have
linear. In contrast, most bacteria have a single, circular four or five genes; the bacterium Escherichia coli has a
(double-stranded) chromosome, although many bacteria single chromosome containing 4,288 genes, and a human
have multiple circular chromosomes and some have linear cell has about 20,000 to 25,000 genes on 46 chromosomes.
chromosomes. The chromosome of E. coli would measure about 1 mm
The chromosomes of all cells are subdivided into basic if unwound and stretched out linearly, and yet this fits
informational packets called genes. A gene can be defined within a cell that measures just over 1 micron across,
from more than one perspective. In classical genetics, the making the stretched-out DNA 1,000 times longer than
term refers to the fundamental unit of heredity responsible the cell (figure 9.3). Still, the bacterial chromosome takes
for a given trait in an organism. In the molecular and bio- up only about one-third to one-half of the cell’s volume.
chemical sense, it is a site on the chromosome that provides Likewise, if the sum of all DNA contained in the 46 hu-
information for a certain cell function. More specifically still, man chromosomes were unraveled and laid end to end,
it is a certain segment of DNA that contains the necessary it would measure about 6 feet. How can such elongated
code to make a protein or RNA molecule. This last definition genomes fit into the minuscule volume of a cell, and in the
of a gene will be emphasized in this chapter. case of eukaryotes, into an even smaller compartment, the
Genes fall into three basic categories: structural genes that nucleus? The answer lies in the regular coiling of the DNA
code for proteins, genes that code for RNA, and regulatory genes chain (see Insight 9.1).
CASE FILE 9 WRAP-UP
The chapter opener described the first known case of infection

with VRSA (in 2002). Analysis of this VRSA strain isolated from
the infected exit site of the patient’s catheter indicated the pos-
sibility that this resistant S. aureus strain had acquired the gene
for vancomycin resistance from the resistant strain of E. faecalis
(VRE), isolated from the patient’s chronic foot ulcer. The fact
that the patient was treated with vancomycin before the VRSA
infection allowed for selection of vancomycin-resistant organ-
isms. Although prior studies showed that the transfer of the
vanA gene by conjugation of Enterococcus species with S. aureus
was possible in vitro (under laboratory conditions), this case
provided good evidence that this gene transfer could occur in
a living patient.
Figure 9.3 An Escherichia coli cell disrupted to release its
See: Staphylococcus aureus resistant to vancomycin—United States,
DNA molecule. 2002. MMWR 51:565–566.
The cell has spewed out its single, uncoiled DNA strand into the Noble, W. C., Virani, Z., and Cree, R. G. 1992. Co-transfer of vancomycin
surrounding medium. and other resistance genes from Enterococcus faecalis NCTC 12201 to
Staphylococcus aureus. FEMS Microbiol. Lett. 93:195–198.
The DNA Code: A Simple

Yet Profound Message between certain bases. Thus, in DNA, the purine adenine
Examining the function of DNA at the molecular level requires (A) always pairs with the pyrimidine thymine (T), and
an even closer look at its structure. To do this we will imagine the purine guanine (G) always pairs with the pyrimidine
being able to magnify a small piece of a gene about 5 million cytosine (C). New research also indicates that the bases are
times. What such fine scrutiny will disclose is one of the great attracted to each other in this pattern because each has a
marvels of biology. James Watson and Francis Crick put the complementary three-dimensional shape that matches its
pieces of the puzzle together in 1953 (Insight 9.1) to discover pair. Although the base-pairing partners generally do not
that DNA is a gigantic molecule, a type of nucleic acid, with vary, the sequence of base pairs along the DNA molecule
two strands combined into a double helix. The general struc- can assume any order, resulting in an infinite number of
ture of DNA is universal, except in some viruses that contain possible nucleotide sequences.
single-stranded DNA. The basic unit of DNA structure is a Other important considerations of DNA structure
nucleotide, and a chromosome in a typical bacterium con- concern the nature of the double helix itself. The halves
sists of several million nucleotides linked end to end. Each are not oriented in the same direction. One side of the
nucleotide is composed of phosphate, deoxyribose sugar, helix runs in the opposite direction of the other, in what
and a nitrogenous base. The nucleotides covalently bond to is called an antiparallel arrangement (figure 9.4b). The order
form a sugar-phosphate linkage that becomes the backbone of the bond between the carbon on deoxyribose and the
of each strand. Each sugar attaches in a repetitive pattern to phosphates is used to keep track of the direction of the
two phosphates. One of the bonds is to the number 5’ (read two sides of the helix. Thus, one helix runs from the 5’ to
“five prime”) carbon on deoxyribose, and the other is to the 3’ direction, and the other runs from the 3’ to 5’ direction.
3’ carbon, which confers a certain order and direction on This characteristic is a significant factor in DNA synthesis
each strand (figure 9.4). and translation.
The nitrogenous bases, purines and pyrimidines, at-
tach by covalent bonds at the 1’ position of the sugar
(figure 9.4a). They span the center of the molecule and The Significance of DNA Structure
pair with appropriate complementary bases from the other
The arrangement of nitrogenous bases in DNA has two
strand. The paired bases are so aligned as to be joined by
essential effects.
hydrogen bonds. Such weak bonds are easily broken, al-
lowing the molecule to be “unzipped” into its complemen- 1. Maintenance of the code during reproduction. The con-
tary strands. This feature is of great importance in gaining stancy of base-pairing guarantees that the code will be
access to the information encoded in the nitrogenous base retained during cell growth and division. When the two
sequence. Pairing of purines and pyrimidines is not ran- strands are separated, each one provides a template (pat-
dom; it is dictated by the formation of hydrogen bonds tern or model) for the replication (exact copying) of a
INSIGHT 9.1 Historical
Deciphering the Structure of DNA

The search for the primary molecules of heredity was a seri- Two English biophysicists, Maurice Wilkins and Rosalind
ous focus throughout the first half of the 20th century. At first, Franklin, had been painstakingly collecting data on X-ray crystal-
many biologists thought that protein was the genetic mate- lographs of DNA for several years. With this technique, molecules
rial. An important milestone occurred in 1944 when Oswald of DNA bombarded by X rays produce a photographic image
Avery, Colin MacLeod, and Maclyn McCarty purified DNA that can predict the three-dimensional structure of the molecule.
and demonstrated at last that it was indeed the blueprint for After being allowed to view certain X-ray data, Watson and Crick
life. This was followed by an avalanche of research, which noticed an unmistakable pattern: The molecule appeared to be a
continues today. double helix. Gradually, the pieces of the puzzle fell into place,
One area of extreme interest concerned the molecular struc- and a final model was assembled—a model that explained all of
ture of DNA. In 1951, American biologist James Watson and the qualities of DNA, including how it is copied. Although Wat-
English physicist Francis Crick collaborated on solving the son and Crick were rightly hailed for the clarity of their solution,
DNA puzzle. Although they did little of the original research, it must be emphasized that their success was due to the consider-
they were intrigued by several findings from other scientists. able efforts of a number of English and American scientists. This
It had been determined by Erwin Chargaff that any model of historic discovery showed that the tools of physics and chemistry
DNA structure would have to contain deoxyribose, phosphate, have useful applications in biological systems, and it also spawned
purines, and pyrimidines arranged in a way that would provide ingenious research in all areas of molecular genetics.
variation and a simple way of copying itself. Watson and Crick Since the discovery of the double helix in 1953, an extensive
spent long hours constructing models with cardboard cutouts body of biochemical, microscopic, and crystallographic analysis
and kept alert for any and every bit of information that might has left little doubt that the model first proposed by Watson and
give them an edge. Crick is correct. Newer techniques using scanning tunneling mi-
croscopy produce three-dimensional images of DNA magnified
2 million times. These images verify the helical shape and twists
of DNA represented by models.
The men who cracked the code of life. Dr. James Watson (left) and The first direct glimpse at DNA’s structure. This false-color scanning
Dr. Francis Crick (right) stand next to their model that finally explained tunneling micrograph of calf thymus gland DNA (2,000,000⫻) brings out
the structure of DNA in 1953. the well-defined folds in the helix.
H
H N O H N H
N G N H N C H
N N
Sugar N H O
Sugar
H
Sugar Nitrogen Sugar

phosphate base pairs phosphate
OH
5′
5´ 3′
P 3′ Base pairs
5′ OH
D G C D
P 5′ P Phosphate
3′
P
T 5′
A
D D 4′ Sugar phosphate
D 1′ Deoxyribose backbone
P 3′ with carbon number
C P 2′
G
D D C Cytosine
P
C P
G G Guanine
D D
P
T P T Thymine
A
D D
P A Adenine
C P
G
D Hydrogen
D
bond
P
T P Covalent
A bond 5′ 3′
5′
3′
D D
3′ (b) (c)
5′
P
P
H
H N N H O CH3
N A N H N T H
N N
Sugar H O
(a)
Figure 9.4 Three views of DNA structure.

(a) A schematic nonhelical model, to show the arrangement of the molecules it is made of. Note that the order of phosphate and sugar bonds
differs between the two strands, going from the #5 carbon to the #3 carbon on one strand, and from the #3 carbon to the #5 carbon on the
other strand. Insets show details of the nitrogen base pairs. (b) Simplified model that highlights the antiparallel arrangement and the major and
minor grooves. (c) Space-filling model that more accurately depicts the three-dimensional structure of DNA.
.. ..
.. ..
.. ..
D
.. .. .. ..
.. .. . ..
.. .. .. D
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
.. Helicase .. .. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
Template
.. strands
.. D ..
.. .. D ..
.. .. .. ..
.. .. .
.. ..
.. ..
.. .. .. ..
.. .. .. ..
.. .. .. ..
. .
(a) (b) (c) (d)
Figure 9.5 Simplified steps to show the semiconservative replication of DNA.

(a, b) The two strands of the double helix are unwound and separated by a helicase, which disrupts the hydrogen bonds and exposes the
nitrogen base codes of DNA. Each single strand formed will serve as a template to synthesize a new strand of DNA. (c) A DNA polymerase
(D) proceeds along the DNA molecule, attaching the correct nucleotides 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. (d) The resultant new DNA molecules contain one strand of the newly
synthesized DNA and the original template strand. The integrity of the code is kept intact because the linear arrangement of the bases is
maintained during this process. Note that the actual details of the process are presented in figure 9.6.
new molecule (figure 9.5). Because the sequence of one different sequences possible. Carried out, this number would
strand automatically gives the sequence of its partner, approximate 1.5 ⫻ 10602, a number so huge that it provides
the code can be duplicated with fidelity. nearly endless degrees of variation.
2. Providing variety. The order of bases along the length
of the DNA strand constitutes the genetic program, or DNA Replication: Preserving the Code
the language, of the DNA code. The message present
in a gene is a precise sequence of these bases, and the
and Passing It On
genome is the collection of all DNA bases that, in an The sequence of bases along the length of a gene constitutes
ordered combination, are responsible for the unique the language of DNA. For this language to be preserved for
qualities of each organism. hundreds of generations, it will be necessary for the genetic
program to be duplicated and passed on to each offspring.
It is tempting to ask how such a seemingly simple code This process of duplication is called DNA replication. In
can account for the extreme differences among forms as the following example, we will show replication in bacteria,
diverse as a virus, E. coli, and a human. The English lan- but with some exceptions, it also applies to the process as
guage, based on 26 letters, can create an infinite variety of it works in eukaryotes and some viruses. Early in binary
words, but how can an apparently complex genetic language fission, the metabolic machinery of a bacterium initiates
such as DNA be based on just four nitrogen base “letters”? the duplication of the chromosome. This DNA replication
A mathematical example can explain the possibilities. For a must be completed during a single generation time (around
segment of DNA that is 1,000 nucleotides long, there are 41,000 20 minutes in E. coli).
The Overall Replication Process the enzyme DNA polymerase III. The entire process of rep-
lication does, however, depend on several enzymes and can
What features allow the DNA molecule to be exactly dupli-
be most easily understood by keeping in mind a few points
cated, and how is its integrity retained? DNA replication
concerning both the structure of the DNA molecule and the
requires a careful orchestration of the actions of 30 different
limitations of DNA polymerase III:
enzymes (partial list in table 9.1), which separate the strands
of the existing DNA molecule, copy its template, and produce 1. The nucleotides that need to be read by DNA polymer-
two complete daughter molecules. A simplified version of ase III are buried deep within the double helix. Accessing
replication is shown in figure 9.5 and includes the following: these nucleotides requires both that the DNA molecule
be unwound and that the two strands of the helix be
1. uncoiling the parent DNA molecule;
separated from one another.
2. unzipping the hydrogen bonds between the base pairs, thus
2. DNA polymerase III is unable to begin synthesizing a
separating the two strands and exposing the nucleotide
chain of nucleotides but can only continue to add nucle-
sequence of each strand (which is normally buried in the
otides to an already existing chain.
center of the helix) to serve as templates; and
3. DNA polymerase III can only add nucleotides in one
3. synthesizing two new strands by attachment of the cor-
direction, so a new strand is always synthesized 5’ to 3’.
rect complementary nucleotides to each single-stranded
template. With these constraints in mind, the details of replication can
be more easily comprehended.
A critical feature of DNA replication is that each daughter
Replication begins when an RNA primer is synthesized
molecule will be identical to the parent in composition, but
and enters at the origin of replication (figure 9.6, step 1).
neither one is completely new; the strand that serves as a
DNA polymerase III cannot begin synthesis unless it has
template is an original parental DNA strand. The preserva-
this short strand of RNA to serve as a starting point for
tion of the parent molecule in this way, termed semiconserv-
adding nucleotides. Because the bacterial DNA molecule is
ative replication, helps explain the reliability and fidelity of
circular, opening of the circle forms two replication forks,
replication.
each containing its own set of replication enzymes. The
DNA polymerase III is a huge enzyme complex that encir-
Refinements and Details of Replication cles the replication fork and adds nucleotides in accordance
The origin of replication is a short sequence rich in adenine with the template pattern. As synthesis proceeds, the forks
and thymine that, you will recall, are held together by only continually open up to expose the template for replication
two hydrogen bonds rather than three. Because the origin (figure 9.6, steps 2, 3).
of replication is AT-rich, less energy is required to separate Because DNA polymerase is correctly oriented for syn-
the two strands than would be required if the origin were thesis only in the 5’ to 3’ direction of the new molecule (red)
rich in guanine and cytosine. Prior to the start of replica- strand, only one strand, called the leading strand, can be
tion, enzymes called helicases (unzipping enzymes) bind to synthesized as a continuous, complete strand. The strand with
the DNA at the origin. These enzymes untwist the helix and the opposite orientation (3’ to 5’) is termed the lagging strand
break the hydrogen bonds holding the two strands together, (figure 9.6, steps 4, 5). Because it cannot be synthesized con-
resulting in two separate strands, each of which will be used tinuously, the polymerase adds nucleotides a few at a time in
as a template for the synthesis of a new strand. the direction away from the fork (5’ to 3’). As the fork opens
The process of synthesizing a new daughter strand of up a bit, the next segment is synthesized backward to the
DNA using the parental strand as a template is carried out by point of the previous segment, a process repeated at both forks
until synthesis is complete. In this way, the DNA polymerase
is able to synthesize the two new strands simultaneously. This
TABLE 9.1 Some Enzymes Involved in DNA manner of synthesis produces one strand containing short
Replication and Their Functions fragments of DNA (100 to 1,000 bases long) called Okazaki
fragments. These fragments are attached to the growing end
Enzyme Function of the lagging strand by another enzyme called DNA ligase.
Helicase Unzipping the DNA helix
Primase Synthesizing an RNA primer Elongation and Termination of the Daughter Molecules
DNA polymerase III Adding bases to the new DNA
The addition of nucleotides proceeds at an astonishing pace,
chain; proofreading the chain estimated in some bacteria to be 750 bases per second at each
for mistakes fork! As replication proceeds, the newly produced double
DNA polymerase I Removing primer, closing gaps, strand loops down (figure 9.7a). The DNA polymerase I re-
repairing mismatches moves the RNA primers used to initiate DNA synthesis and
Ligase Final binding of nicks in DNA replaces them with DNA. When the forks come full circle and
during synthesis and repair meet, ligases move along the lagging strand to begin the initial
Gyrase Supercoiling linking of the fragments and to complete synthesis and sepa-
ration of the two circular daughter molecules (figure 9.7b).
Origin of replication
Primer molecules
1 Replication origin. Short

RNA primers are positioned
to start replication.
DNA III polymerase complexes
3′ 5′
2 Strands separate;
two polymerase complexes
attach at origin. Arrows 5′ 3′
indicate direction of
replication. 5 In actuality, a loop forms in the lagging
Replication forks
strand so that the portion of the lagging
strand undergoing replication is oriented
in the same direction as the leading
3′ 5′–3′ 5′ strand (i.e., both strands are parallel).
3 At primer sequence, This allows the polymerase complex to
each polymerase complex move along both strands in the 5′ to 3′
synthesizes two strands prime direction at the same time,
5′ 3′–5′ 3′ synthesizing both strands simultaneously.
at the replication forks.
Details of the process are seen in the inset,
although, for purposes of clarity, the loop
is not shown.
Lagging strands Leading strands 5′
5′ Template
3′
3′ strand
3′ 5′ Leading 5′
5′ 3′ strand
4 Since DNA polymerase acts 3′
only in the 5′ to 3′ direction, 3′ 5′
it forms a continuous leading Okazaki 5′
5′ Lagging fragments
strand from that orientation. 3′ strand
5′ 3′ 3′
The lagging strand, which
orients 3′ to 5′, must be Template
5′
made backward in short strand
sections, 5′ to 3′, which are
later linked together. Note Polymerase
that the numbers refer to the 3′ complex
direction of synthesis of the
new strand (red).
Process Figure 9.6 The bacterial replicon: a model for DNA synthesis.
1 Circular DNA has a special origin site where replication originates. 2 When strands are separated, two replication forks form, and a DNA
polymerase III complex enters at each fork. 3 Starting at the primer sequence, each polymerase moves along the template strands (blue),
synthesizing the new strands (red) at each fork. 4 DNA polymerase works only in the 5‘ to 3‘ direction, necessitating a different pattern of
replication at each fork. Because the leading strand orients in the 5‘ to 3‘ direction, it will be synthesized continuously. The lagging strand, which
orients in the opposite direction, can only be synthesized in short sections, 5‘ to 3‘, which are later linked together. 5 Inset presents details of
process at one replication fork and shows the Okazaki fragments and the relationship of the template, leading, and lagging strands.
Like any language, DNA is occasionally “misspelled” and can lead to serious cell dysfunction and even death.
when an incorrect base is added to the growing chain. Because continued cellular integrity is very dependent on
Studies have shown that such mistakes are made once in accurate replication, cells have evolved their own proofread-
approximately 108 to 109 bases, but most of these are cor- ing function for DNA. DNA polymerase III, the enzyme that
rected. If not corrected, they are referred to as mutations elongates the molecule, can also detect incorrect, unmatching
9.2 Applications of the DNA Code: Transcription and Translation 255
Their DNA is packaged in tightly wound spirals arranged

Forks
in discrete chromosomes.
■ DNA copies itself just before cellular division by the process
of semiconservative replication. Semiconservative replica-
tion means that each “old” DNA strand is the template upon
(a) 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 synthesized—one continuously and one in short
fragments—and mistakes are proofread and removed.
Nick
9.2 Applications of the DNA Code:

Transcription and Translation
We have explored how the genetic message in the DNA mol-
ecule is conserved through replication. Now we must consider
the precise role of DNA in the cell. 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 by the cell? Although the genome
is full of critical information, the molecule itself does not per-
form cell processes directly. Its stored information is conveyed
to RNA molecules, which carry out instructions. The concept
that genetic information flows from DNA to RNA to protein
Daughter cell Daughter cell
is a central theme of molecular biology (figure 9.8a). More
(b) precisely, it states that the master code of DNA is first used to
Figure 9.7 Completion of chromosome replication synthesize an RNA molecule via a process called transcrip-
in bacteria. tion, and the information contained in the RNA is then used
(a) As replication proceeds, one double strand loops down. (b) Final to produce proteins in a process known as translation. The
separation is achieved through repair and the release of two completed principal exceptions to this pattern are found in RNA viruses,
molecules. The daughter cells receive these during binary fission. which convert RNA to other RNA, and in retroviruses, which
convert RNA to DNA.
bases; excise them; and replace them with the correct base. This “central dogma,” which outlined the primary
DNA polymerase I can also proofread the molecule and re- understanding of genetics during the first half century of the
pair damaged DNA. genetic revolution (since the 1950s), has very recently been
shown to be incomplete. While it is true that proteins are
Replication in Other Biological Systems The replication made in accordance with this central dogma, there is more to
pattern of eukaryotes is similar to that of prokaryotes. It also the story (figure 9.8b). In addition to the RNA that is used to
uses a variety of DNA polymerases, and replication proceeds produce proteins, a wide variety of RNAs are used to regulate
in both directions from the point of origin. gene function. Many of the genetic malfunctions that cause
human disease are in fact found in these regulatory RNA seg-
ments—and not in genes for proteins as was once thought.
■ CHECKPOINT The DNA that codes for these very crucial RNA molecules
was called “junk” DNA until very recently. We say more
■ Nucleic acids are molecules that contain the blueprints of life about this in Insight 9.2.
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 Gene-Protein Connection
■ The total amount of DNA in an organism is termed its genome
(also genotype). The genome of each species contains a unique The Triplet Code and the Relationship to Proteins
arrangement of genes that define its appearance (phenotype), Several questions invariably arise concerning the relationship
metabolic activities, and pattern of reproduction. between genes and cell function. For instance, how does gene
■ The genome of prokaryotes is quite small compared with structure lead to the expression of traits in the individual, and
the genome of eukaryotes. Bacterial DNA consists of a few what features of gene expression cause one organism to be so
thousand genes in one circular chromosome. Eukaryotic
distinctly different from another? For answers, we must turn to
genomes range from thousands to tens of thousands of genes.
the correlation between gene and protein structure. We know
Triplets Single nucleotide

1 2 3 4 5
DNA
DNA
(a) (b) Codon

1 2 3 4 5
mRNA
Transcription of DNA (copy of
one strand)
Regulatory RNAs
Amino acids
1 2 3 4 5
tRNA mRNA rRNA
Variations in the order and types

Translation of RNA will dictate the shape
and function of the protein.
Ribosome
(rRNA + protein) Figure 9.9 Simplified view of the DNA-protein
Antisense RNA, relationship.
micro RNA, The DNA molecule is a continuous chain of base pairs, but the
tRNA interfering RNA, and sequence must be interpreted in groups of three base pairs (a
riboswitches that
regulate transcription triplet). Each triplet as copied into mRNA codons will translate into
and translation one amino acid; consequently, the ratio of base pairs to amino
mRNA acids is 3:1.
2. Proteins ultimately determine phenotype, the expression

Protein
of all aspects of cell function and structure. Put more sim-
ply, living things are what their proteins make them. Reg-
Expression of DNA ulatory RNAs help determine which proteins are made.
for structure and 3. DNA is mainly a blueprint that tells the cell which kinds
functions of cell
of proteins and RNAs to make and how to make them.
Figure 9.8 Summary of the flow of genetic

information in cells. The Major Participants in Transcription
DNA is the ultimate storehouse and distributor of genetic information. and Translation
(a) DNA must be deciphered into a usable cell language. It does this
by transcribing its code into RNA helper molecules that translate Transcription, the formation of RNA using DNA as a tem-
that code into protein. (b) Other sections of the DNA produce very plate, and translation, the synthesis of proteins using RNA
important RNA molecules that regulate genes and their products. as a template, are highly complex. A number of components
participate: most prominently, messenger RNA, transfer
RNA, regulatory RNAs, ribosomes, several types of enzymes,
that each structural gene is a linear sequence of nucleotides that
and a storehouse of raw materials. After first examining each
codes for a protein. Because each protein is different, each gene
of these components, we shall see how they come together in
must also differ somehow in its composition. In fact, the lan-
the assembly line of the cell.
guage of DNA exists in the order of groups of three consecu-
tive bases called triplets on one DNA strand (figure 9.9). Thus,
one gene differs from another in its composition of triplets. An RNAs: Tools in the Cell’s Assembly Line
equally important part of this concept is that each triplet repre- Ribonucleic acid is an encoded molecule like DNA, but its
sents a code for a particular amino acid. When the triplet code general structure is different in several ways:
is transcribed and translated, it dictates the type and order of
1. It is a single-stranded molecule that exists in helical form.
amino acids in a polypeptide (protein) chain.
This single strand can assume secondary and tertiary
The final key points that connect DNA and an organism’s
levels of complexity due to bonds within the molecule,
traits are:
leading to specialized forms of RNA (tRNA and rRNA—
1. A protein’s primary structure—the order and type of see figure 9.8a).
amino acids in the chain—determines its characteristic 2. RNA contains uracil, instead of thymine, as the comple-
shape and function. mentary base-pairing mate for adenine. This does not
INSIGHT 9.2 Discovery
Small RNAs: An Old Dog Shows Off Some New(?) Tricks

Since the earliest days of molecular biology, RNA has been an strand of the DNA that produces mRNA. This antisense molecule
overlooked worker of the cell, quietly ferrying the information in has the ability to pair with the “sense,” or messenger, RNA and
DNA to ribosomes to direct the formation of proteins. Current re- thus keep it from being transcribed. Riboswitches, RNAs that at-
search however is showing a new, dynamic role for RNA in the cell tach to a chemical with one end and only then become available
that may forever change the reputation of this humble molecule. for translation on the other end, were isolated for the first time in
Short lengths of RNA seem to have the ability to control the 2002. One riboswitch has been found to regulate the expression of
expression of certain genes. Some of these are called micro RNAs 26 important genes in the bacterium Bacillus subtilis. Riboswitches
and some are called small interfering RNAs. They do this by have probably been around since the early days of life on the
folding back on themselves after being transcribed, and by doing planet. So, although they are new to us, they have been used to
so they activate a system inside cells that degrades dsRNA. Cells regulate gene expression for billions of years.
do this in order to rid themselves of invading viruses (which These newly discovered RNA molecules have answered some
are organisms that might have dsRNA). The micro RNAs also vexing questions that came out of the genome sequencing studies
bind with mRNA of certain genes, thereby causing them to be (led by the Human Genome Project). Most of the DNA in organ-
degraded as well. The repressing nature of dsRNA was discov- isms was found not to code for functional proteins. In humans, the
ered quite accidentally when researchers were trying to induce “junk” percentage was 98%! Yet, in bacteria as well as humans, the
expression of genes by providing them in dsRNA form; instead, junk DNA was preserved in the same form for the last millions of
genes matching those RNA sequences were shut down entirely years of evolution, suggesting it had a very important function.
through this clever regulatory system. In 2006, the Nobel Prize We now know that much of this “junk” DNA codes for these im-
for Medicine or Physiology was awarded to the two American portant RNA regulatory molecules.
scientists, Andrew Fire and Craig Mello, who discovered this The RNA regulatory molecules are being heavily exploited to
phenomenon. accomplish research tasks that were never before possible. More
A second type of regulation seems to occur when small RNAs important, molecules such as antisense RNA are being explored
alter the structure of chromosomes. As DNA and proteins coil for their therapeutic uses in cases where defective genes need to
together to form chromatin, small RNAs direct how tightly or be shut down in order to restore a patient to health.
loosely the chromatin is constructed. Just as a closed book cannot Our knowledge of the full role of small RNAs in the cell is just
be read, DNA sequences contained within tightly coiled chroma- beginning. In the meantime, scientists will keep studying small
tin are generally inaccessible to the cell, silencing the expression RNAs while being mindful of the old adage, “Good things come
of those genes. Antisense RNA is produced from the opposite in small packages.”
change the inherent DNA code in any way because the The many functional types of RNA range from small regula-
uracil still follows the pairing rules. tory pieces to large structural ones (table 9.2 and Insight 9.2).
3. Although RNA, like DNA, contains a backbone that con- All types of RNA are formed through transcription of a DNA
sists of alternating sugar and phosphate molecules, the gene, but only mRNA is further translated into another type
sugar in RNA is ribose rather than deoxyribose. of molecule (protein).
TABLE 9.2 Types of Ribonucleic Acid

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 Forms the major part of a ribosome and No
molecules participates in protein synthesis
Micro (miRNA) antisense, Regulatory RNAs Regulation of gene expression and coiling No
riboswitch, and small of chromatin
interfering (siRNA)
Primer An RNA that can begin DNA Primes DNA No
replication
Ribozymes RNA enzymes, parts of splicer enzymes Remove introns from other RNAs in eukaryotes No
3⬘
Amino acid 5⬘
attachment site A 5⬘
(a) Transfer RNA (tRNA). C G 3⬘
Transfer RNA (tRNA) can loop G C
back on itself to form intrachain C G
hydrogen bonds. The result of the Amino acid
U G H bonds attachment site
secondary structure is a cloverleaf
structure, shown here in simplified U A
form. At its bottom is an anticodon A U
that specifies the attachment of a C A U
particular amino acid at the 3⬘ end. U
C C G U G
At right is a three-dimensional view A A C CA
G A U
of the tertiary structure of tRNA.
C G U G U C G
T C G A G
U A G G
G AG C
Hairpin C
G
loops
U A
C
G
AC
A
Anticodon U
U C
A Anticodon
C U G A U G A C U
(b) Messenger RNA (mRNA).
A short piece of messenger R R R R R R R R R
RNA (mRNA) illustrates the
general structure of RNA: P P P P P P P P
single strandedness,
repeating phosphate-ribose
sugar backbone attached to
Codon 1 Codon 2 Codon 3
single nitrogen bases; use
of uracil instead of thymine.
P = Phosphate R = Ribose U = Uracil
Figure 9.10 Characteristics of transfer and messenger RNA.
Messenger RNA: Carrying DNA’s Message This compact molecule is an adaptor that converts RNA
language into protein language. The bottom loop of the clo-
Messenger RNA (mRNA) is a transcript (copy) of a structural
verleaf exposes a triplet, the anticodon, that both designates
gene or genes in the DNA. It is synthesized by a process similar
the specificity of the tRNA and complements mRNA’s co-
to synthesis of the leading strand during DNA replication, and
dons. At the opposite end of the molecule is a binding site for
the complementary base-pairing rules ensure that the code will
the amino acid that is specific for that tRNA’s anticodon. For
be faithfully copied in the mRNA transcript. The message of
each of the 20 amino acids, there is at least one specialized
this transcribed strand is later read as a series of triplets called
type of tRNA to carry it. Binding of an amino acid to its spe-
codons (figure 9.10), and the length of the mRNA molecule
cific tRNA, a process known as “charging” the tRNA, takes
varies from about 100 nucleotides to several thousand. The de-
place in two enzyme-driven steps: First an ATP activates the
tails of transcription and the function of mRNA in translation
amino acid, and then this group binds to the acceptor end of
will be covered shortly.
the tRNA. Because tRNA is the molecule that will convert
the master code on mRNA into a protein, the accuracy of this
Transfer RNA: The Key to Translation step is crucial.
Transfer RNA (tRNA) is also a copy of a specific region of
DNA; however, it differs from mRNA. It is uniform in length,
being 75 to 95 nucleotides long, and it contains sequences of
The Ribosome: A Mobile Molecular
bases that form hydrogen bonds with complementary sec- Factory for Translation
tions of the same tRNA strand. At these points, the mol- The prokaryotic (70S) ribosome is a particle composed of
ecule bends back upon itself into several hairpin loops, giving tightly packaged ribosomal RNA (rRNA) and protein. The
the molecule a secondary cloverleaf structure that folds even rRNA component of the ribosome is also a long polynucleotide
further into a complex, three-dimensional helix (figure 9.10). molecule. It forms complex three-dimensional figures that
1 Overall view of a gene. RNA polymerase binding site

Each gene contains a Leader Initiation Termination
specific promoter region sequence codon sequences
and a leader sequence for
guiding the beginning of Promoter
transcription. This is region ( (
followed by the region of T A C G A C T G A T G C
the gene that codes for a A T G C T G A C T A C G(
polypeptide and ends with (
a series of terminal
RNA Intervening sequence of variable size
sequences that stop
translation. polymerase Template strand Termination sequence
3⬘ 5⬘
2 DNA is unwound at
the promoter by RNA
polymerase. Only one
strand of DNA, called
the template strand,
is copied by the RNA 5⬘ 3⬘
polymerase. This Coding strand
Unwinding of DNA
strand runs in the 3⬘
to 5⬘ direction.
Direction of
3 As the RNA transcription
polymerase moves
along the strand, it
adds
complementary
nucleotides as 3⬘
dictated by the
DNA template,
forming the single- Nucleotide
stranded mRNA 5⬘ pool
that reads in the 5⬘
Early mRNA
to 3⬘ direction.
transcript
Elongation
4 The polymerase
continues transcribing
until it reaches a
termination site and 3⬘
the mRNA transcript
is released for
translation. Note that
the section of the
5⬘
DNA that has been
transcribed is Late mRNA transcript
rewound into its
original configuration.
Process Figure 9.11 The major events in mRNA synthesis (transcription).
contribute to the structure and function of ribosomes. The The nontranscribed strand is called the coding strand. The
interactions of proteins and rRNA create the two subunits strand of DNA that serves as a template varies from one gene
of the ribosome that engage in final translation of the genetic to another.
code (see figure 9.12). A metabolically active bacterial cell can Transcription is initiated when RNA polymerase recog-
accommodate up to 20,000 of these minuscule factories—all nizes a segment of the DNA called the promoter region. This
actively engaged in reading the genetic program, taking region consists of two sequences of DNA just prior to the
in raw materials, and producing proteins at an impressive beginning of the gene to be transcribed. The first sequence,
rate. which occurs approximately 35 bases prior to the start of
transcription, is tightly bound by RNA polymerase. Tran-
Transcription: The First Stage scription is allowed to begin when the DNA helix begins to
unwind at the second sequence, which is located about 10
of Gene Expression bases prior to the start of transcription. As the DNA helix
During transcription, the DNA code is converted to RNA unwinds, the polymerase advances and begins synthesizing
through several stages, directed by a huge and very complex an RNA molecule complementary to the template strand of
enzyme system, RNA polymerase (figure 9.11). Only one DNA. The nucleotide sequence of promoters differs only
strand of the DNA—the template strand—contains meaning- slightly from gene to gene, with all promoters being rich in
ful instructions for synthesis of a functioning polypeptide. adenine and thymine.
these molecules and stabilizes reactions between them. The

Large small subunit binds to the 5’ end of the mRNA, and the large
Amino acids subunit
subunit supplies enzymes for making peptide bonds on the
protein. The ribosome begins to scan the mRNA by moving
Exit site
P A in the 5’ to 3’ direction along the mRNA. The first codon
it encounters is the START codon, which is almost always
E AUG (and, rarely, GUG).
With the mRNA message in place on the assembled
Small
ribosome, the next step in translation involves entrance
subunit of tRNAs with their amino acids. The pool of cytoplasm
contains a complete array of tRNAs, previously charged by
5⬘ having the correct amino acid attached. The step in which
the complementary tRNA meets with the mRNA code is
tRNAs guided by the two sites on the large subunit of the ribos-
mRNA ome called the P site (left) and the A site (right).1 Think
transcript
of these sites as shallow depressions in the larger subunit
Figure 9.12 The “players” in translation. of the ribosome, each of which accommodates a tRNA.
A ribosome serves as the stage for protein synthesis. Assembly of the The ribosome also has an exit or E site where used tRNAs
small and large subunits results in specific sites for holding the mRNA are released.
and two tRNAs with their amino acids.
The Master Genetic Code:

During elongation, which proceeds in the 5’ to 3’ direc-
tion (with regard to the growing RNA molecule), the mRNA
The Message in Messenger RNA
is assembled by the addition of nucleotides that are comple- By convention, the master genetic code is represented by the
mentary to the DNA template. Remember that uracil (U) is mRNA codons and the amino acids they specify (figure 9.13).
placed as adenine’s complement. As elongation continues, Except in a very few cases, this code is universal, whether for
the part of DNA already transcribed is rewound into its prokaryotes, eukaryotes, or viruses. It is worth noting that
original helical form. At termination, the polymerases recog- once the triplet code on mRNA is known, the original DNA
nize another code that signals the separation and release of sequence, the complementary tRNA code, and the types of
the mRNA strand, also called the transcript. How long is the amino acids in the protein are automatically known (fig-
mRNA? The very smallest mRNA might consist of 100 bases; ure 9.14). However, one cannot predict (backward) from pro-
an average-size mRNA might consist of 1,200 bases; and a tein structure what the exact mRNA codons are because of a
large one might consist of several thousand. factor called redundancy,2 meaning that a particular amino
acid can be coded for by more than a single codon.
In figure 9.13, the mRNA codons and their correspond-
Translation: The Second Stage ing amino acid specificities are given. Because there are 64
of Gene Expression different triplet codes3 and only 20 different amino acids, it
In translation, all of the elements needed to synthesize a is not surprising that some amino acids are represented by
protein, from the mRNA to the amino acids, are brought several codons. For example, leucine and serine can each be
together on the ribosomes (figure 9.12). The process occurs in represented by any of six different triplets, and only tryp-
five stages: initiation, elongation, termination, protein fold- tophan and methionine are represented by a single codon.
ing, and protein processing. In such codons as leucine, only the first two nucleotides are
required to encode the correct amino acid, and the third nu-
cleotide does not change its sense. This property, called wob-
Initiation of Translation ble, is thought to permit some variation or mutation without
The mRNA molecule leaves the DNA transcription site and altering the message.
is transported to ribosomes in the cytoplasm. Ribosomal sub-
units are specifically adapted to assembling and forming sites
to hold the mRNA and tRNAs. The ribosomes of prokaryotes The Beginning of Protein Synthesis
and eukaryotes are different sizes. Prokaryotic ribosomes, With mRNA serving as the guide, the stage is finally set for
as well as the ribosomes in chloroplasts and mitochondria actual protein assembly. The correct tRNA (labeled 1 on
of eukaryotes, are of a 70s size, made up of a 50s (large)
subunit and a 30s (small) subunit. The “s” is a measurement
of sedimentation rates, which is how ribosomes are charac- 1. P stands for peptide site; A stands for aminoacyl (amino acid) site; E stands
terized. It is a nonlinear measure; therefore, 30s and 50s add for exit site.
up to 70s. Eukaryotic ribosomes are 80s (a large subunit of 2. This property is also called “degeneracy” by some books.
60s and a 40s small subunit). The ribosome thus recognizes 3. 64 ⫽ 43 (the four different codons in all possible combinations of three).
Second Base Position

U C A G
U
UUU
UUC } Phenylalanine
UCU
UCC
Serine
UAU
UAC } Tyrosine
UGU
UGC } Cysteine
U
C
UUA
UUG } Leucine
UCA
UCG
UAA
UAG
} STOP**
UGA
UGG
STOP**
Tryptophan
A
G
CUU
CUC
CCU
CCC
CAU
CAC } Histidine
CGU
CGC
U
C
C Leucine Proline Arginine
Third Base Position

First Base Position
CUA
CUG
CCA
CCG
CAA
CAG } Glutamine
CGA
CGG
A
G
AUU
AUC
Isoleucine
ACU
ACC
AAU
AAC } Asparagine
AGU
AGC } Serine
U
C
A Threonine
AUA
AUG START fMethionine*
ACA
ACG
AAA
AAG } Lysine
AGA
AGG } Arginine
A
G
}
GUU GCU GAU GGU U
Aspartic acid
GUC GCC GAC GGC C
G Valine Alanine Glycine
GUA
GUG
GCA
GCG
GAA
GAG } Glutamic acid
GGA
GGG
A
G
* This codon initiates translation.

**For these codons, which give the orders to stop translation, there are no corresponding tRNAs and no amino acids.
Figure 9.13 The genetic code: codons of mRNA that specify a given amino acid.
The master code for translation is found in the mRNA codons.
DNA
Coding strand
figure 9.15) enters the P site and binds to the start codon
triplets C T
G (AUG) presented by the mRNA. Rules of pairing dictate that
A T C
T
A
G G A A C G the anticodon of this tRNA must be complementary to the
A C G T
T A mRNA codon AUG; thus, the tRNA with anticodon UAC
C T G C
will first occupy site P. It happens that the amino acid carried
Template strand by the initiator tRNA in bacteria is formyl methionine (fMet;
G C U
mRNA A U C
U A see figure 9.13), though, in many cases, it may not remain a
codons G A C G permanent part of the finished protein.
tRNA UAC GA C UGA UGC
anticodons
Continuation and Completion of Protein
Synthesis: Elongation and Termination
While reviewing the dynamic process of protein assembly,
Protein
(amino you will want to remain aware that the ribosome shifts its
acids “reading frame” to the right along the mRNA from one codon
F-Methionine
Leucine
Threonine
Threonine
specified) to the next. This brings the next codon into place on the ribos-
ome and makes a space for the next tRNA to enter the A posi-
tion. A peptide bond is formed between the amino acids on
the adjacent tRNAs, and the polypeptide grows in length.
Same amino acid; has a Elongation begins with the filling of the A site by a sec-
different codon and anticodon ond tRNA (2 on figure 9.15). The identity of this tRNA and its
Figure 9.14 Interpreting the DNA code. amino acid is dictated by the second mRNA codon.
If the DNA sequence is known, the mRNA codon can be surmised. If a The entry of tRNA 2 into the A site brings the two
codon is known, the anticodon and, finally, the amino acid sequence adjacent tRNAs in favorable proximity for a peptide bond to
can be determined. The reverse is not possible (determining the form between the amino acids (aa) they carry. The fMet is
exact codon or anticodon from amino acid sequence) due to the transferred from the first tRNA to aa 2, resulting in two cou-
redundancy of the code. pled amino acids called a dipeptide (figure 9.15, step 2).
Leucine Peptide bond 2

1 3
f Met 2 2
1
A
P A
P E
2
E
1 2 3
G C
A GCU G C G C
Anticodon U AC C UG C CG AU
A G
G C UG C CG
A UG
AUC
mRNA G CU
Codon A
UC
UA G U
AG
1 Entrance of tRNAs 1 and 2
5 Formation of peptide bond
Peptide bond 1
4
2 Alanine
1
A
P
2 1
E 1 2
3 4
G C
A
GCU
U AC C UG C CG 2 C
GA
A UG
AUC
C
GA P
E 3
UA G
2 Formation of peptide bond G

GC GC U AU C
CC G
1 AUG C U G
Empty tRNA 2 UA G
1
A
6 Discharge of tRNA 2; second
translocation; enter tRNA 4
C
UA
P 2
E
G AC Peptide bond 3
GCU
C UG C CG
A UG 4
AUC
1 2
3
A
P site UA G P
E 4
3 Discharge of tRNA 1 at E site 3 Repeat to
C A stop codon
3 G
G C GC U A UC
G
Proline CC G
AUG C U G
UA G Stop codon
1 3
2 7 Formation of peptide bond
G C
G
E P
A
2
G C
A
AU C UG C CG
G
GC
U A
UC
A site UA
G
4 First translocation; tRNA 2 shifts into

P site; enter tRNA 3 by ribosome Process Figure 9.15 The events in protein synthesis.
For the next step to proceed, some room must be made after the codon for the last amino acid. Termination codons—
on the ribosome, and the next codon in sequence must be UAA, UAG, and UGA—are codons for which there is no cor-
brought into position for reading. This process is accom- responding tRNA. Although they are often called nonsense
plished by translocation, the enzyme-directed shifting of codons, they carry a necessary and useful message: Stop here.
the ribosome to the right along the mRNA strand, which When this codon is reached, a special enzyme breaks the
causes the blank tRNA (1) to be discharged from the ri- bond between the final tRNA and the finished polypeptide
bosome (figure 9.15, step 3) at the E site. This also shifts chain, releasing it from the ribosome.
the tRNA holding the dipeptide into P position. Site A is Before newly made proteins can carry out their struc-
temporarily left empty. The tRNA that has been released tural or enzymatic roles, they often require finishing touches.
is now free to drift off into the cytoplasm and become re- Even before the peptide chain is released from the ribosome,
charged with an amino acid for later additions to this or it begins folding upon itself to achieve its biologically active
another protein. tertiary conformation. Other alterations, called posttransla-
The stage is now set for the insertion of tRNA 3 at site A tional modifications, may be necessary. Some proteins must
as directed by the third mRNA codon (figure 9.15, step 4). have the starting amino acid (formyl methionine) clipped off;
This insertion is followed once again by peptide bond forma- proteins destined to become complex enzymes have cofac-
tion between the dipeptide and aa 3 (making a tripeptide), tors added; and some join with other completed proteins to
splitting of the peptide from tRNA 2, and translocation. This form quaternary levels of structure.
releases tRNA 2, shifts mRNA to the next position, moves The operation of transcription and translation is ma-
tRNA 3 to position P, and opens position A for the next chinelike in its precision. Protein synthesis in bacteria is both
tRNA (which will be called tRNA 4). From this point on, efficient and rapid. At 37°C, 12 to 17 amino acids per second
peptide elongation proceeds repetitively by this same series are added to a growing peptide chain. An average protein
of actions out to the end of the mRNA. consisting of about 400 amino acids requires less than half a
The termination of protein synthesis is not simply a mat- minute for complete synthesis. Further efficiency is gained
ter of reaching the last codon on mRNA. It is brought about when the translation of mRNA starts while transcription is
by the presence of at least one special codon occurring just still occurring (figure 9.16). A single mRNA is long enough
mRNA RNA polymerase
Transcription
Start of
translation
(a)
Growing
polypeptides
3 Polyribosomal
complex
4
Start
7
5 6
(b) (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.
to be fed through more than one ribosome DNA E I E I E I E

simultaneously. This permits the synthesis template
of hundreds of protein molecules from the
same mRNA transcript arrayed along a chain Exon Intron
of ribosomes. This polyribosomal complex Primary
is indeed an assembly line for mass produc- mRNA E I E I E I E
tion of proteins. Protein synthesis consumes transcript
an enormous amount of energy. Nearly 1,200
ATPs are required just for synthesis of an
average-size protein. Lariat forming Spliceosomes
Occurs
in
nucleus Transcript
processed
Eukaryotic Transcription by special
E E E E
and Translation: Similar enzymes

Yet Different Lariat excised
Eukaryotes and prokaryotes share many simi-
larities in protein synthesis. The start codon in
Spliceosomes released Exons
eukaryotes is also AUG, but it codes for a dif- spliced
ferent form of methionine. Another difference together
is that eukaryotic mRNAs code for just one E E E E
protein, unlike bacterial mRNAs, which of-
ten contain information from several genes Occurs
in series. in
There are a few differences between cytoplasm mRNA transcript can
now be translated
prokaryotic and eukaryotic gene expression.
The presence of the DNA in a separate com- Figure 9.17 The split gene of eukaryotes.
partment (the nucleus) means that eukaryotic Eukaryotic genes have an additional complicating factor in their translation. Their
transcription and translation cannot be si- coding sequences, or exons (E), are interrupted at intervals by segments called introns
multaneous. The mRNA transcript must pass (I) that are not part of that protein’s code. Introns are transcribed but not translated,
through pores in the nuclear membrane and which necessitates their removal by RNA splicing enzymes before translation.
be carried to the ribosomes in the cytoplasm
for translation.
We have given the simplified definition of a gene that enzyme loops the introns into lariat-shaped pieces, excises
works well for prokaryotes, but most eukaryotic genes them, and joins the exons end to end. By this means, a
(and, surprisingly, archaeal genes) do not exist as an unin- strand of mRNA with no intron material is produced. This
terrupted series of triplets coding for a protein. A eukaryo- completed mRNA strand can then proceed to the cyto-
tic gene contains the code for a protein, but located along plasm to be translated.
the gene are one to several intervening sequences of bases, Several different types of introns have been discovered,
called introns, that do not code for protein. Introns are in- some of which do code for cell substances. As detailed in In-
terspersed between coding regions, called exons, that will sight 9.2, a great deal of non-protein-coding DNA is proving
be translated into protein (figure 9.17). We can use words to be vital for cell function. In humans, this intron material
as examples. A short section of colinear prokaryotic gene represents 98% of the DNA and the discovery of its extreme
might read TOM SAW OUR DOG DIG OUT; a eukaryotic importance has revolutionized the “genetic revolution.”
gene that codes for the same portion would read TOM SAW Another surprising finding from 2006 was the discovery of
XZKP FPL OUR DOG QZWVP DIG OUT. The recognizable proteins that had sections that were in reverse order from
words are the exons, and the nonsense letters represent the DNA sequence that encoded them. Previously it was
the introns. considered a hard and fast rule that DNA sequence deter-
This unusual genetic architecture, sometimes called mined the mRNA and then the amino acid sequence once
a split gene, requires further processing before transla- the introns were accounted for. This new data revealed that
tion. Transcription of the entire gene with both exons and cellular machinery can flip stretches of amino acids around,
introns occurs first, producing a pre-mRNA. A series of essentially creating new proteins from the same gene. Many
adenosines is added to the mRNA molecule. This protects introns have been found to code for an enzyme called re-
the molecule and eventually directs it out of the nucleus verse transcriptase, which can convert RNA into DNA.
for translation. Next, a type of RNA and protein called a Other introns are translated into endonucleases, enzymes
spliceosome recognizes the exon-intron junctions and en- that can snip DNA and allow insertions and deletions into
zymatically cuts through them. The action of this splicer the sequence.
9.3 Genetic Regulation of Protein Synthesis and Metabolism 265
The Genetics of Animal Viruses 9.3 Genetic Regulation of Protein

The genetic nature of viruses was described in chapter 6. Synthesis and Metabolism
Viruses essentially consist of one or more pieces of DNA or
RNA enclosed in a protective coating. Above all, they are In chapter 8, we surveyed the metabolic reactions in cells
genetic parasites that require access to their host cell’s genetic and the enzymes involved in those reactions. At that time,
and metabolic machinery to be replicated, transcribed, and we mentioned that some enzymes are regulated and that one
translated; and they also have the potential for genetically form of regulation occurs at the genetic level. Control mecha-
changing the cells. Because they contain only those genes nisms ensure that genes are active only when their products
needed for the production of new viruses, the genomes of are required. In this way, enzymes will be produced as they
viruses tend to be very compact and economical. In fact, this are needed and prevent the waste of energy and materials
very simplicity makes them excellent subjects for the study in dead-end synthesis. Antisense RNAs, micro RNAs, and
of gene function. riboswitches (see Insight 9.2) provide regulation in both
The genetics of viruses is quite diverse. In many vi- prokaryotes and eukaryotes. Prokaryotes have an additional
ruses, the nucleic acid is linear in form; in others, it is strategy: they organize collections of genes into operons.
circular. The genome of most viruses exists in a single mol- Operons consist of a coordinated set of genes, all of which
ecule, though in a few, it is segmented into several smaller are regulated as a single unit. Operons are described as either
molecules. Most viruses contain normal double-stranded inducible or repressible. The category each operon falls into
(ds) DNA or single-stranded (ss) RNA, but other patterns is determined by how transcription is affected by the envi-
exist. There are ssDNA viruses, dsRNA viruses, and ret- ronment surrounding the cell. Many catabolic operons are
roviruses, which work backward by making dsDNA from inducible, meaning that the operon is turned on (induced)
ssRNA. In some instances, viral genes overlap one another, by the substrate of the enzyme for which the structural genes
and in a few DNA viruses, both strands contain a translat- code. In this way, the enzymes needed to metabolize a nutri-
able message. ent (lactose, for example) are only produced when that nutri-
A few generalities can be stated about viral genetics. In ent is present in the environment. Repressible operons often
all cases, the viral nucleic acid penetrates the cell and is in- contain genes coding for anabolic enzymes, such as those
troduced into the host’s gene-processing machinery at some used to synthesize amino acids. In the case of these operons,
point. In successful infection, an invading virus instructs the several genes in series are turned off (repressed) by the prod-
host’s machinery to synthesize large numbers of new virus uct synthesized by the enzyme.
particles by a mechanism specific to a particular group. With
few exceptions, replication of the DNA molecule of DNA
animal viruses occurs in the nucleus, where the cell’s DNA The Lactose Operon: A Model for
replication machinery lies and the genome of RNA viruses Inducible Gene Regulation in Bacteria
is replicated in the cytoplasm. In all viruses, viral mRNA is The best understood cell system for explaining control
translated into viral proteins on host cell ribosomes using through genetic induction is the lactose (lac) operon. This
host tRNA. system, first described in 1961 by François Jacob and Jacques
Monod, accounts for the regulation of lactose metabolism in
Escherichia coli. Many other operons with similar modes of
■ CHECKPOINT action have since been identified, and together they furnish
convincing evidence that the environment of a cell can have
■ Information in DNA is converted to proteins by the process of
great impact on gene expression.
transcription and translation. These proteins may be structural
or functional in nature. Structural proteins contribute to the The lactose operon has three important features (fig-
architecture of the cell while functional proteins (enzymes) ure 9.18):
control an organism’s metabolic activities. 1. the regulator, composed of the gene that codes for a pro-
■ The DNA code occurs in groups of three bases; this code is tein capable of repressing the operon (a repressor);
copied onto RNA as codons; the message determines the
2. the control locus, composed of two areas, the promoter
types of amino acids in a protein. This code is universal in
(recognized by RNA polymerase) and the operator, a se-
all cells and viruses.
quence that acts as an on/off switch for transcription; and
■ DNA also contains a great number of non-protein-coding
sequences. These sequences are often transcribed into RNA 3. the structural locus, made up of three genes, each coding
that serves to regulate cell function. for a different enzyme needed to catabolize lactose.
■ The processes of transcription and translation are similar One of the enzymes, ␤-galactosidase, hydrolyzes the lactose
but not identical for prokaryotes and eukaryotes. Eukary- into its monosaccharides; another, permease, brings lactose
otes transcribe DNA in the nucleus, remove its introns, and
across the cell membrane.
translate it in the cytoplasm. Bacteria transcribe and translate
The operon provides an efficient strategy that permits
simultaneously because the DNA is not sequestered in a
nucleus and the bacterial DNA is free of introns. genes for a particular metabolic pathway to be induced or
repressed in unison by a single regulatory element. The
(a) Operon Off. In the absence of lactose, a R e g ulat o r Rep Repressor

repressor protein (the product of a regulatory
gene located elsewhere on the bacterial ion
RNA polymerase ript ural ge
ne 3
chromosome) attaches to the operator of the nsc truct
Tra S
operon. This effectively locks the operator and en e2
g
prevents any transcription of structural genes ral
u ctu
downstream (to its right). Suppression of S tr
e1
Re
n
transcription (and consequently, of translation) al ge
P r o m oter Structur
p
prevents the unnecessary synthesis of Operator
enzymes for processing lactose.
Locked Translation
Lactose (inducer)
l gene 3
ctura
(b) Operon On. Upon entering the cell, the
2 S tr u
substrate (lactose) becomes a genetic inducer n e
l ge
by attaching to the repressor, which loses its ct ura
S tr u
grip and falls away. The RNA polymerase is
n e1
now free to bind to the promoter and initiate al ge
P r o m oter Operator Structur
transcription, and the enzymes produced by
translation of the mRNA perform the necessary
reactions on their lactose substrate.
ne 3
tion ural ge
RNA polymerase rip S truct
nsc e2
active Tra g en
ral
u ctu
S tr
n e1
al ge
mRNA
Transcription
into enzymes
Inactive
repressor
Lactose
transported
and digested
Figure 9.18 The lactose operon in bacteria: how inducible genes are controlled by substrate.
enzymes of the lac operon are of the inducible sort men- region and is transcribed constitutively because it is not
tioned in chapter 8. The promoter, operator, and structural controlled in tandem with the operon.
components lie adjacent to one another, but the regulator can If lactose is added to the cell’s environment, it triggers
be at a distant site. several events that turn the operon on. The binding of lactose
In inducible systems like the lac operon, the operon to the repressor protein causes a conformational change in
is normally in an off mode and does not initiate enzyme the repressor that dislodges it from the operator segment
synthesis when the appropriate substrate is absent (fig- (figure 9.18b). With the operator opened up, RNA polymer-
ure 9.18a). How is the operon maintained in this mode? The ase can now bind to the promoter. The structural genes are
key is in the repressor protein that is coded by the regulatory transcribed in a single unbroken transcript coding for all
gene. This relatively large molecule is allosteric, meaning it three enzymes. (During translation, however, each protein
has two binding sites, one for the operator and another for is synthesized separately.) Because lactose is ultimately re-
lactose. In the absence of lactose, this repressor binds with sponsible for stimulating protein synthesis, it is called an
the operator locus, thereby blocking the transcription of the inducer.
structural genes lying downstream. Think of the repressor As lactose is depleted, further enzyme synthesis is not
as a lock on the operator, and if the operator is locked, the necessary, so the order of events reverses. At this point,
structural genes cannot be transcribed. Importantly, the there is no longer sufficient lactose to inhibit the repressor;
regulator gene lies upstream (to the left) of the operator hence the repressor is again free to attach to the operator.
9.3 Genetic Regulation of Protein Synthesis and Metabolism 267
(a) Operon On. A repressible operon remains

n l gene 3
tio ctura on when its nutrient products (here, arginine)
RNA polymerase crip S tr u
ns ene
2 are in great demand by the cell because the
Tra g
tu ral repressor is unable to bind to the operator at
St r uc low nutrient levels.
n e1
al ge
Repressor is inactive
(wrong shape to attach
to operator).
Rep Enzymes
synthesize
arginine.
Arginine immediately
used in metabolism
(b) Operon Off. The operon is repressed

when (1) arginine builds up and, serving as a
l gene 3 corepressor, activates the repressor. (2) The
ctura
2 Stru repressor complex affixes to the operator and
en e
g blocks the RNA polymerase and further
tu ral
r uc transcription of genes for arginine synthesis.
St
n e1
al ge
Arginine
accumulates.
Repressor is active
(1) (correct shape
achieved).
RNA ral g ene 3

ked ructu
polymerase
n bloc e 2 St
io n
ipt l ge
scr ura sted
Tran S tr
u ct
s a r re
1 s i
ne he
Structur
al ge ynt
(2) P r o m oter Operator i n es
i n
Arg
Figure 9.19 Repressible operon: control of a gene through excess nutrient.
The operator is locked, and transcription of the structural different principle—that of repression. Similar factors such
genes and enzyme synthesis related to lactose both stop. as repressor proteins, operators, and a series of structural
A fine but important point about the lac operon is that it genes exist for this operon but with some important differ-
functions only in the absence of glucose or if the cell’s energy ences. Unlike the lac operon, this operon is normally in the
needs are not being met by the available glucose. Glucose is on mode and will be turned off only when this nutrient is no
the preferred carbon source because it can be used immedi- longer required. The excess nutrient serves as a corepressor
ately in growth and does not require induction of an operon. needed to block the action of the operon.
When glucose is present, a second regulatory system ensures A growing cell that needs the amino acid arginine (arg)
that the lac operon is inactive, regardless of lactose levels in effectively illustrates the operation of a repressible operon.
the environment. Under these conditions, the arg operon is set to on and ar-
ginine is being actively synthesized through the action of the
operon’s enzymatic products (figure 9.19a). In an active cell,
A Repressible Operon the arginine will be used immediately, and the repressor will
Bacterial systems for synthesis of amino acids, purines and remain inactive (unable to bind the operator) because there is
pyrimidines, and many other processes work on a slightly too little free arginine to activate it. As the cell’s metabolism
begins to slow down, however, the synthesized arginine will

no longer be used up and will accumulate. The free arginine
■ CHECKPOINT
is then available to act as a corepressor by attaching to the ■ Gene expression must be orchestrated to coordinate the
repressor. This reaction changes the shape of the repressor, organism’s needs with nutritional resources. Genes can be
making it capable of binding to the operator. Transcription turned “on” and “off” by specific molecules, which expose
stops; arginine is no longer synthesized (figure 9.19b). or hide their nucleotide codes for transcribing proteins. Most,
In eukaryotic cells, gene function can be altered by but not all, of these proteins are enzymes.
intrinsic regulatory segments similar to operons. Some ■ Operons are collections of genes in bacteria that code for
products with a coordinated function. They include genes
molecules, called transcription factors, insert on the grooves
for operational and structural components of the cell. Nutri-
of the DNA molecule and enhance transcription of specific
ents can combine with regulator gene products to turn a set
genes. Examples include zinc “fingers” and leucine “zip- of structural genes on (inducible genes) or off (repressible
pers.” These transcription factors can regulate gene expres- genes). The lac (lactose) operon is an example of an inducible
sion in response to environmental stimuli such as nutrients, operon. The arg (arginine) operon is an example of a repres-
toxin levels, or even temperature. Eukaryotic genes are also sible operon.
regulated during growth and development, leading to the ■ The rifamycins, tetracyclines, and aminoglycosides are classes
hundreds of different tissue types found in higher multicel- of antibiotics that are effective because they interfere with
lular organisms. transcription and translation processes in microorganisms.
Antibiotics That Affect Transcription

and Translation
Naturally occurring cell nutrients are not the only agents
9.4 Mutations: Changes
capable of modifying gene expression. Some infection in the Genetic Code
therapy is based on the concept that certain drugs react
with DNA, RNA, or ribosomes and thereby alter genetic As precise and predictable as the rules of genetic expression
expression (see chapter 12). Treatment with such drugs is seem, permanent changes do occur in the genetic code. In-
based on an important premise: that growth of the infectious deed, genetic change is the driving force of evolution. In mi-
agent will be inhibited by blocking its protein-synthesizing croorganisms, such changes may become evident in altered
machinery selectively, without disrupting the cell synthesis gene expression, such as the appearance or disappearance of
of the patient receiving the therapy. anatomical or physiological traits. For example, a pigmented
Drugs that inhibit protein synthesis exert their influence bacterium can lose its ability to form pigment, or a strain of
on transcription or translation. For example, the rifamycins the malarial parasite can develop resistance to a drug. When
used in therapy for tuberculosis bind to RNA polymerase, phenotypic changes are due to changes in the genotype, it is
blocking the initiation step of transcription, and are selec- called a mutation. On a strictly molecular level, a mutation
tively more active against bacterial RNA polymerase than is an alteration in the nitrogen base sequence of DNA. It can
the corresponding eukaryotic enzyme. Actinomycin D binds involve the loss of base pairs, the addition of base pairs, or
to bacterial DNA and halts mRNA chain elongation, but it a rearrangement in the order of base pairs. Do not confuse
also binds to human DNA. For this reason, it is very toxic this with genetic recombination, in which microbes transfer
and never used to treat bacterial infections, though it can whole segments of genetic information between themselves.
be applied in tumor treatment. A microorganism that exhibits a natural, nonmutated
The ribosome is a frequent target of antibiotics that in- characteristic is known as a wild type, or wild strain. If a
hibit ribosomal function and ultimately protein synthesis. microorganism bears a mutation, it is called a mutant strain.
The value and safety of these antibiotics again depend upon Mutant strains can show variance in morphology, nutri-
the differential susceptibility of prokaryotic and eukaryotic tional characteristics, genetic control mechanisms, resistance
ribosomes. One problem with drugs that selectively disrupt to chemicals, temperature preference, and nearly any type
prokaryotic ribosomes is that the mitochondria of humans of enzymatic function. Mutant strains are very useful for
contain a prokaryotic type of ribosome, and these drugs may tracking genetic events, unraveling genetic organization, and
inhibit the function of the host’s mitochondria. One group pinpointing genetic markers. A classic method of detecting
of antibiotics (including erythromycin and spectinomycin) mutant strains involves addition of various nutrients to a
prevents translation by interfering with the attachment of culture to screen for its use of that nutrient. For example,
mRNA to ribosomes. Chloramphenicol, lincomycin, and in a culture of a wild-type bacterium that is lactose-positive
tetracycline bind to the ribosome in a way that blocks the (meaning it has the necessary enzymes for fermenting this
elongation of the polypeptide, and aminoglycosides (such as sugar), a small number of mutant cells have become lactose-
streptomycin) inhibit peptide initiation and elongation. It is negative, having lost the capacity to ferment this sugar. If the
interesting to note that these drugs have served as important culture is plated on a medium containing indicators for fer-
tools to explore genetic events because they can arrest spe- mentation, each colony can be observed for its fermentation
cific stages in these processes. reaction and the negative strain isolated. Another standard
9.4 Mutations: Changes in the Genetic Code 269
Culture of bacteria TABLE 9.3 Selected Mutagenic Agents

with discrete colonies and Their Effects
Agent Effect
Plate exposed to a Chemical
mutagenic agent
Nitrous acid, bisulfite Removes an amino group
from some bases
Ethidium bromide Inserts between the
paired bases
Acridine dyes Cause frameshifts due to
insertion between
Special velveteen replica base pairs
plating carrier (sterile)
picks up tiny bits of colony
Nitrogen base analogs Compete with natural
when pressed upon agar bases for sites on
and removed. replicating DNA
Radiation
Ionizing (gamma rays, Form free radicals that
X rays) cause single or double
breaks in DNA
Pressed down
Ultraviolet Causes cross-links between
adjacent pyrimidines
Medium that
selects for Nonselective
mutants medium carefully controlled use of mutagens has proved a useful
way to induce mutant strains of microorganisms for study.
Chemical mutagenic agents act in a variety of ways to
change the DNA. Agents such as acridine dyes insert com-
pletely across the DNA helices between adjacent bases to
produce mutations that distort the helix. Analogs4 of the
nitrogen bases (5-bromodeoxyuridine and 2-aminopurine,
for example) are chemical mimics of natural bases that are
incorporated into DNA during replication. Addition of these
Colonies of Mixture of wild-type abnormal bases leads to mistakes in base-pairing. Many
mutant strains and mutant strains chemical mutagens are also carcinogens, or cancer-causing
Figure 9.20 The general basis of replica plating. agents (see the discussion of the Ames test in a later section
This method was developed by Joshua Lederberg for detecting and of this chapter).
isolating mutant strains of microorganisms. Physical agents that alter DNA are primarily types of
radiation. High-energy gamma rays and X rays introduce ma-
jor physical changes into DNA, and it accumulates breaks
method of detecting and isolating microbial mutants is by that may not be repairable. Ultraviolet (UV) radiation induces
replica plating (figure 9.20). abnormal bonds between adjacent pyrimidines that prevent
normal replication. Exposure to large doses of radiation can be
Causes of Mutations fatal, which is why radiation is so effective in microbial con-
trol; it can also be carcinogenic in animals. (The use of UV to
A mutation is described as spontaneous or induced, control microorganisms is described further in chapter 11.)
depending upon its origin. A spontaneous mutation is a
random change in the DNA arising from errors in replication
that occur randomly. The frequency of spontaneous muta- Categories of Mutations
tions has been measured for a number of organisms. Mu- Mutations range from large mutations, in which large ge-
tation rates vary tremendously, from one mutation in 105 netic sequences are gained or lost, to small ones that affect
replications (a high rate) to one mutation in 1010 replications only a single base on a gene. These latter mutations, which
(a low rate). The rapid rate of bacterial reproduction allows involve addition, deletion, or substitution of single bases, are
these mutations to be observed more readily in bacteria than called point mutations.
in most eukaryotes.
Induced mutations result from exposure to known mu-
tagens, which are primarily physical or chemical agents that 4. An analog is a chemical structured very similarly to another chemical
interact with DNA in a disruptive manner (table 9.3). The except for minor differences in functional groups.
To understand how a change in DNA influences the cell, TABLE 9.4 Classification of Major Types
remember that the DNA code appears in a particular order of of Mutations
triplets (three bases) that is transcribed into mRNA codons,
each of which specifies an amino acid. A permanent alteration Example
in the DNA that is copied faithfully into mRNA and trans- (a) Wild type THE BIG BAD DOG ATE THE FAT
lated can change the structure of the protein. A change in a (original, nonmutated RED CAT
protein can likewise change the morphology and physiology sequence)
of a cell. Some mutations have a harmful effect on the cell,
Substitution mutations
leading to cell dysfunction or death; these are called lethal
mutations. Neutral mutations produce neither adverse nor (b) Missense THE BIG BAD DOG ATE THE FIT
RED CAT
helpful changes. A small number of mutations are beneficial
in that they provide the cell with a useful change in structure (c) Nonsense THE BIG BAD (stop)
or physiology. Frameshift mutations
Any change in the code that leads to placement of a dif- (d) Insertion THE BIG BAB DDO GAT ETH
ferent amino acid is called a missense mutation. A missense EFA
mutation can do one of the following: TRE DCA T
(e) Deletion THE BIG BDD OGA TET HEF ATR
1. create a faulty, nonfunctional (or less functional) protein,
EDC AT
2. produce a protein that functions in a different manner, or
3. cause no significant alteration in protein function. Categories of mutations based on type of DNA alteration
(a) The wild-type sequence of a gene is the DNA sequence
A nonsense mutation, on the other hand, changes a
found in most organisms and is generally considered the
normal codon into a stop codon that does not code for an “normal” sequence. (b) A missense mutation causes a different
amino acid and stops the production of the protein wher- amino acid to be incorporated into a protein. Effects range from
ever it occurs. A nonsense mutation almost always results unnoticeable to severe, based on how different the two amino
in a nonfunctional protein. A silent mutation alters a base acids are. (c) A nonsense mutation converts a codon to a stop
but does not change the amino acid and thus has no effect. codon, resulting in premature termination of protein synthesis.
For example, because of the redundancy of the code, ACU, Effects of this type of mutation are almost always severe. (d, e)
ACC, ACG, and ACA all code for threonine, so a mutation Insertion and deletion mutations cause a change in the reading
that changes only the last base will not alter the sense of the frame of the mRNA, resulting in a protein in which every amino
message in any way. A back-mutation occurs when a gene acid after the mutation is affected. Because of this, frameshift
mutations almost always result in a nonfunctional protein.
that has undergone mutation reverses (mutates back) to its
original base composition.
Mutations also occur when one or more bases are in-
serted into or deleted from a newly synthesized DNA
strand. This type of mutation, known as a frameshift, is repair mechanism requires visible light and a light-sensitive
so named because the reading frame of the mRNA has enzyme, DNA photolyase, which can detect and attach to
been changed. Frameshift mutations nearly always result the damaged areas (sites of abnormal pyrimidine bind-
in a nonfunctional protein because every amino acid after ing). Ultraviolet repair mechanisms are successful only for
the mutation is different from what was coded for in the a relatively small number of UV mutations. Cells cannot
original DNA. Also note that insertion or deletion of bases repair severe, widespread damage and will die. In humans,
in multiples of three (3, 6, 9, etc.) results in the addition or the genetic disease xeroderma pigmentosa is due to nonfunc-
deletion of amino acids but does not disturb the reading tioning genes for the enzyme photolyase. Persons suffering
frame. The effects of all of these types of mutations can be from this rare disorder develop severe skin cancers; this
seen in table 9.4. relation provides strong evidence for a link between cancer
and mutations.
Mutations can be excised by a series of enzymes that
Repair of Mutations remove the incorrect bases and add the correct ones. This
Earlier we indicated that DNA has a proofreading mechanism process is known as excision repair. First, enzymes break the
to repair mistakes in replication that might otherwise become bonds between the bases and the sugar-phosphate strand
permanent (see page 254). Because mutations are potentially at the site of the error. A different enzyme subsequently
life-threatening, the cell has additional systems for finding removes the defective bases one at a time, leaving a gap that
and repairing DNA that has been damaged by various mu- will be filled in by DNA polymerase I and ligase (figure 9.21).
tagenic agents and processes. Most ordinary DNA damage A repair system can also locate mismatched bases that were
is resolved by enzymatic systems specialized for finding and missed during proofreading: for example, C mistakenly
fixing such defects. paired with A, or G with T. The base must be replaced soon
DNA that has been damaged by ultraviolet radiation after the mismatch is made, or it will not be recognized by
can be restored by photoactivation or light repair. This the repair enzymes.
9.4 Mutations: Changes in the Genetic Code 271
Enzyme complex I One indicator organism in the Ames test is a mutant

strain of Salmonella typhimurium6 that has lost the ability
to synthesize the amino acid histidine, a defect highly sus-
ceptible to back-mutation because the strain also lacks DNA
repair mechanisms. Mutations that cause reversion to the
wild strain, which is capable of synthesizing histidine, occur
Removed spontaneously at a low rate. A test agent is considered a mu-
tagen if it enhances the rate of back-mutation beyond levels
that would occur spontaneously. One variation on this testing
procedure is outlined in figure 9.22. The Ames test has proved
(a)
invaluable for screening an assortment of environmental and
dietary chemicals for mutagenicity and carcinogenicity with-
Enzyme complex II out resorting to animal studies.
Added
Positive and Negative Effects of Mutations
Many mutations are not repaired. How the cell copes with
them depends on the nature of the mutation and the strategies
available to that organism. Mutations are permanent and
heritable and will be passed on to the offspring of organisms
and new viruses and become a long-term part of the gene
pool. Most mutations are harmful to organisms; others pro-
(b) vide adaptive advantages.
If a mutation leading to a nonfunctional protein occurs
in a gene for which there is only a single copy, as in haploid
or simple organisms, the cell will probably die. This happens
when certain mutant strains of E. coli acquire mutations in
the genes needed to repair damage by UV radiation. Muta-
tions of the human genome affecting the action of a single
protein (mostly enzymes) are responsible for more than 3,500
diseases (see chapter 10).
Although most spontaneous mutations are not beneficial,
(c)
a small number contribute to the success of the individual
Figure 9.21 Excision repair of mutation by enzymes. and the population by creating variant strains with alter-
(a) The first enzyme complex recognizes one or several incorrect bases nate ways of expressing a trait. Microbes are not “aware” of
and removes them. (b) The second complex (DNA polymerase I and this advantage and do not direct these changes; they simply
ligase) places correct bases and seals the gaps. (c) Repaired DNA. respond to the environment they encounter. Those organisms
with beneficial mutations can more readily adapt, survive,
and reproduce. In the long-range view, mutations and the
variations they produce are the raw materials for change in
The Ames Test the population and, thus, for evolution.
New agricultural, industrial, and medicinal chemicals are Mutations that create variants occur frequently enough
constantly being added to the environment, and exposure that any population contains mutant strains for a number of
to them is widespread. The discovery that many such com- characteristics, but as long as the environment is stable, these
pounds are mutagenic and that up to 83% of these mutagens mutants will never comprise more than a tiny percentage of
are linked to cancer is significant. Although animal testing the population. When the environment changes, however,
has been a standard method of detecting chemicals with it can become hostile for the survival of certain individuals,
carcinogenic potential, a more rapid screening system called and only those microbes bearing protective mutations will
the Ames test5 is also commonly used. In this ingenious test, be equipped to survive in the new environment. In this way,
the experimental subjects are bacteria whose gene expres- the environment naturally selects certain mutant strains that
sion and mutation rate can be readily observed and moni- will reproduce, give rise to subsequent generations, and in
tored. The premise is that any chemical capable of mutating time, be the dominant strain in the population. Through these
bacterial DNA can similarly mutate mammalian (and thus means, any change that confers an advantage during selection
human) DNA and is therefore potentially hazardous.
6. S. typhimurium inhabits the intestine of poultry and causes food poisoning

5. Named for its creator, Bruce Ames. in humans. It is used extensively in genetic studies of bacteria.
Culture of Salmonella bacteria,

histidine(–)
In the control setup, bacteria The eperimental plate is

are plated on a histidine-free prepared the same way
medium containing liver except that it contains the test
enzymes but lacking the test agent. After incubation, plates
agent. (a) Control Plate (b) Test Plate are observed for colonies.
Minimal medium Minimal medium Any colonies developing on
with no histidine with test chemical the plates are due to a
and no test chemical and no histidine back-mutation in a cell, which
has reverted it to a
Incubation (12 h) histidine(+) strain.
Any colonies that form have
back-mutated to histidine(+).
Histidine(+) colonies arising from Histidine(+) colonies induced

spontaneous back-mutation by the chemical
(c) The degree of mutagenicity of the chemical agent can be

calculated by comparing the number of colonies growing on the
control plate with the number on the test plate. Chemicals that
produce an increased incidence of back-mutation are
considered carcinogens.
Figure 9.22 The Ames test.

This test is based on a strain of Salmonella typhimurium that cannot synthesize histidine [his(⫺)]. It lacks the enzymes to repair DNA so that
mutations show up readily, and it has leaky cell walls that permit the ready entrance of chemicals. Many potential carcinogens (benzanthracene
and aflatoxin, for example) are mutagenic agents only after being acted on by mammalian liver enzymes, so an extract of these enzymes is
added to the test medium.
pressure will be retained by the population. One of the clear- donates DNA to another bacterium is a type of genetic trans-
est models for this sort of selection and adaptation is acquired fer termed recombination, the end result of which is a new
drug resistance in bacteria (see chapter 12). Bacteria have also strain different from both the donor and the original recipi-
developed a mechanism for increasing their adaptive capacity ent strain. Recombination in bacteria depends in part on the
through genetic exchange, called genetic recombination. fact that bacteria contain extrachromosomal DNA—that is,
plasmids—and are adept at interchanging genes. Genetic
exchanges have tremendous effects on the genetic diversity
9.5 DNA Recombination Events of bacteria. They provide additional genes for resistance to
drugs and metabolic poisons, new nutritional and metabolic
Genetic recombination through sexual reproduction is an im- capabilities, and increased virulence and adaptation to the
portant means of genetic variation in eukaryotes. Although environment.
bacteria have no exact equivalent to sexual reproduction, In general, any organism that contains (and expresses)
they exhibit a primitive means for sharing or recombining genes that originated in another organism is called a recom-
parts of their genome. An event in which one bacterium binant.
9.5 DNA Recombination Events 273
Transmission of Genetic Material role in conjugation is denoted by F⫹ for the cell that has the
in Bacteria F plasmid and by F⫺ for the cell that lacks it. Contact is made
when a pilus grows out from the F⫹ cell, attaches to the sur-
DNA transfer between bacterial cells typically involves face of the F⫺ cell, contracts, and draws the two cells together
small pieces of DNA in the form of plasmids or chromo- (figure 9.23a; see also figure 4.8). In both gram-positive and
somal fragments. Plasmids are small, circular pieces of DNA gram-negative cells, an opening is created between the con-
that contain their own origin of replication and therefore can nected cells, and the replicated DNA passes across from one
replicate independently of the bacterial chromosome. Plas- cell to the other (figure 9.23b). Conjugation is a conservative
mids are found in many bacteria (as well as some fungi) and process, in that the donor bacterium generally retains a copy
typically contain, at most, only a few dozen genes. Although of the genetic material being transferred.
plasmids are not necessary for bacterial survival, they often There are hundreds of conjugative plasmids with some
carry useful traits, such as antibiotic resistance. Chromo- variations in their properties. One of the best understood
somal fragments that have escaped from a lysed bacterial plasmids is the F factor in E. coli, which exhibits these pat-
cell are also commonly involved in the transfer of genetic terns of transfer:
information between cells. An important difference between
plasmids and fragments is that while a plasmid has its own 1. The donor (F⫹) cell makes a copy of its F factor and trans-
origin of replication and is stably replicated and inherited, mits this to a recipient (F⫺) cell. The F⫺ cell is thereby
chromosomal fragments must integrate themselves into the changed into an F⫹ cell capable of producing a pilus and
bacterial chromosome in order to be replicated and eventu- conjugating with other cells (figure 9.23c). No additional
ally passed to progeny cells. The process of genetic recom- donor genes are transferred at this time.
bination is rare in nature, but its frequency can be increased 2. In high-frequency recombination (Hfr) donors, the fertility
in the laboratory, where the ability to shuffle genes between factor has been integrated into the F⫹ donor chromosome.
organisms is highly prized. The term high-frequency recombination was adopted to
Depending upon the mode of transmission, the means of denote that a cell with an integrated F factor transmits its
genetic recombination in bacteria is called conjugation, trans- chromosomal genes at a higher frequency than other cells.
formation, or transduction. Conjugation requires the attach- The F factor can direct a more comprehensive transfer
ment of two related species and the formation of a bridge of part of the donor chromosome to a recipient cell. This
that can transport DNA. Transformation entails the transfer transfer occurs through duplication of the DNA by, after
of naked DNA and requires no special vehicle. Transduction which one strand of DNA is retained by the donor, and the
is DNA transfer mediated through the action of a bacterial other strand is transported across to the recipient cell (fig-
virus (table 9.5). ure 9.23d). The F factor may not be transferred during this
process. The transfer of an entire chromosome takes about
Conjugation: Bacterial “Sex” 100 minutes, but the pilus bridge between cells is ordinarily
Conjugation is a mode of genetic exchange in which a plas- broken before this time, and rarely is the entire genome of
mid or other genetic material is transferred by a donor to the donor cell transferred.
a recipient cell via a direct connection (figure 9.23). Both Conjugation has great biomedical importance. Special
gram-negative and gram-positive cells can conjugate. In resistance (R) plasmids, or factors, that bear genes for resist-
gram-negative cells, the donor has a plasmid (fertility, or F’, ing antibiotics and other drugs are commonly shared among
factor) that allows the synthesis of a conjugative pilus. The bacteria through conjugation. Transfer of R factors can con-
recipient cell has a recognition site on its surface. A cell’s fer multiple resistance to antibiotics such as tetracycline,
TABLE 9.5 Types of Intermicrobial Exchange

Examples of
Mode Factors Involved Direct or Indirect* Genes Transferred
Conjugation Donor cell with pilus Direct Drug resistance; resistance to

Fertility plasmid in donor metals; toxin production;
Both donor and recipient alive enzymes; adherence molecules;
Bridge forms between cells to transfer DNA. degradation of toxic substances;
uptake of iron
Transformation Free donor DNA (fragment) Indirect Polysaccharide capsule; unlimited
Live, competent recipient cell with cloning techniques
Transduction Donor is lysed bacterial cell. Indirect Toxins; enzymes for sugar
Defective bacteriophage is carrier of donor DNA. fermentation; drug resistance
Live recipient cell of same species as donor
*Direct means the donor and recipient are in contact during exchange; indirect means they are not.
Physical F Factor Transfer Hfr Transfer

Conjugation
Bridge
Donor Recipient Donor Recipient
F+ F– F factor
Integration of F factor
F factor into chromosome
(plasmid) Hfr
cell Pilus
Pilus
Chromosome
Chromosome
Partial copy of
F+ F– donor chromosome
(a) The pilus of donor cell
(top) attaches to receptor on
recipient cell and retracts to F factor
draw the two cells together. being
copied
Bridge broken Donated genes

F+ F+
F factor
(b) An opening or pore forms (c) Transfer of the F factor, or (d) High-frequency (Hfr) transfer involves
between the cell walls, thereby conjugative plasmid transmission of chromosomal genes from
creating a bridge to transmit a donor cell to a recipient cell. The donor
genetic material. chromosome is duplicated and transmitted
in part to a recipient cell, where it is
integrated into the chromosome.
Figure 9.23 Conjugation: genetic transmission through direct contact between two cells.
chloramphenicol, streptomycin, sulfonamides, and penicil- have a rough (R) appearance and are nonvirulent. (Recall
lin. This phenomenon is discussed further in chapter 12. from chapter 4 that the capsule protects a bacterium from
Other types of R factors carry genetic codes for resistance to the phagocytic host defenses.) To set the groundwork, Griffith
heavy metals (nickel and mercury) or for synthesizing viru- showed that when mice were injected with a live, virulent
lence factors (toxins, enzymes, and adhesion molecules) that (S) strain, they soon died (figure 9.24a). Mice injected with
increase the pathogenicity of the bacterial strain. Conjuga- a live, nonvirulent (R) strain remained alive and healthy
tion studies have also provided an excellent way to map the (figure 9.24b). Next he tried a variation on this theme. First,
bacterial chromosome. he heat-killed an S strain and injected it into mice, which
remained healthy (figure 9.24c). Then came the ultimate test:
Griffith injected both dead S cells and live R cells into mice,
Transformation: Capturing DNA from Solution with the result that the mice died from pneumococcal blood
One of the cornerstone discoveries in microbial genetics was infection (figure 9.24d). If killed bacterial cells do not come
made in the late 1920s by the English biochemist Frederick back to life and the nonvirulent live strain was harmless,
Griffith working with Streptococcus pneumoniae and labora- why did the mice die? Although he did not know it at the
tory mice. The pneumococcus exists in two major strains time, Griffith had demonstrated that dead S cells, while pass-
based on the presence of the capsule, colonial morphology, ing through the body of the mouse, broke open and released
and pathogenicity. Encapsulated strains have a smooth (S) some of their DNA (by chance, that part containing the genes
colonial appearance and are virulent; strains lacking a capsule for making a capsule). A few of the live R cells subsequently
Strain of Cell Type Effect

Colony Strain of Cell Type Effect
Capsule Colony
No capsule
Smooth (S) Live S Survives

strain
Rough (R) Live R
strain
Dies
(a) (b)
Live R strain
Survives
Heat-killed
S strain
Heat-killed
S strain Live S and R strains Dies
isolated from dead
(c) (d) mouse
Figure 9.24 Griffith’s classic experiment in transformation.

In essence, this experiment proved that DNA released from a killed cell can be acquired by a live cell. The cell receiving this new DNA is
genetically transformed—in this case, from a nonvirulent strain to a virulent one.
picked up this loose DNA and were transformed by it into bacteria also exchange genes for antibiotic resistance and
virulent, capsule-forming strains. bacteriocin synthesis in this way.
Later studies supported the concept that a chromosome Because transformation requires no special appendages
released by a lysed cell breaks into fragments small enough and the donor and recipient cells do not have to be in direct
to be accepted by a recipient cell and that DNA, even from contact, the process is useful for certain types of recombinant
a dead cell, retains its genetic code. This nonspecific accept- DNA technology. With this technique, foreign genes from
ance by a bacterial cell of small fragments of soluble DNA a completely unrelated organism are inserted into a plas-
from the surrounding environment is termed transformation. mid, which is then introduced into a competent bacterial
Transformation is apparently facilitated by special DNA- cell through transformation. These recombinations can be
binding proteins on the cell wall that capture DNA from the carried out in a test tube, and human genes can be experi-
surrounding medium. Cells that are capable of accepting mented upon and even expressed outside the human body
genetic material through this means are termed competent. by placing them in a microbial cell. This same phenomenon
The new DNA is processed by the cell membrane and trans- in eukaryotic cells, termed transfection, is an essential aspect
ported into the cytoplasm, where some of it is inserted into of genetically engineered yeasts, plants, and mice, and it has
the bacterial chromosome. Transformation is a natural event been proposed as a future technique for curing genetic dis-
found in several groups of gram-positive and gram-negative eases in humans. These topics are covered in more detail in
bacterial species. In addition to genes coding for the capsule, chapter 10.
Transduction: The Case of the Piggyback DNA Phage DNA

Bacteriophages (bacterial viruses) have been previously Donor (host) chromosome
described as destructive bacterial parasites. Viruses can in
1
fact serve as genetic vectors (an entity that can bring foreign
DNA into a cell). The process by which a bacteriophage
serves as the carrier of DNA from a donor cell to a recipient Cell A (donor)
cell is transduction. Although it occurs naturally in a broad
spectrum of bacteria, the participating bacteria in a single
transduction event must be the same species because of the Parts of phage
Separated piece of host DNA
specificity of viruses for host cells.
There are two versions of transduction. In generalized 2
transduction (figure 9.25), random fragments of disintegrat-
ing host DNA are taken up by the phage during assembly.
Virtually any gene from the bacterium can be transmitted
through this means. In specialized transduction (figure 9.26),
a highly specific part of the host genome is regularly in- Newly assembled phage
incorporating piece
corporated into the virus. This specificity is explained by of host DNA
the prior existence of a temperate prophage inserted in a
fixed site on the bacterial chromosome. When activated, the
prophage DNA separates from the bacterial chromosome,
carrying a small segment of host genes with it. During a
lytic cycle, these specific viral-host gene combinations are
incorporated into the viral particles and carried to another Lysis
bacterial cell.
Several cases of specialized transduction have bio- 3
medical importance. The virulent strains of bacteria such
as Corynebacterium diphtheriae, Clostridium spp., and Strep-
tococcus pyogenes all produce toxins with profound physio-
DNA from donor
logical effects, whereas nonvirulent strains do not produce
toxins. It turns out that toxicity arises from the expres-
sion of bacteriophage genes that have been introduced by 4
transduction. Only those bacteria infected with a temper-
ate phage are toxin formers. (Details of toxin action are
Cell B (recipient)
discussed in the organ-system-specific disease chapters.)
Another instance of transduction is seen in staphylococcal Incorporated into chromosome
transfer of drug resistance.
5
Transposons: “This Gene Is Jumpin’” One type of genetic
transferral of great interest involves transposable elements,
Cell survives and utilizes transduced DNA.
or transposons. Transposons have the distinction of shifting
from one part of the genome to another and so are termed Process Figure 9.25 Generalized transduction:
“jumping genes.” When the idea of their existence in corn genetic transfer by means of a virus carrier.
plants was first postulated by geneticist Barbara McClintock 1 A phage infects cell A (the donor cell) by normal means.
in 1951, it was greeted with nearly universal skepticism 2 During replication and assembly, a phage particle incorporates
because it had long been believed that the location of a a segment of bacterial DNA by mistake. 3 Cell A then lyses and
given gene was set and that genes did not or could not move releases the mature phages, including the genetically altered one.
around. Now it is evident that jumping genes are widespread 4 The altered phage adsorbs to and penetrates another host cell
(cell B), injecting the DNA from cell A rather than viral nucleic acid.
among prokaryotic and eukaryotic cells and viruses.
5 Cell B receives this donated DNA, which recombines with its own
All transposons share the general characteristic of traveling
DNA. Because the virus is defective (biologically inactive as a virus),
from one location to another on the genome—from one chro- it is unable to complete a lytic cycle. The transduced cell survives and
mosomal site to another, from a chromosome to a plasmid, or can use this new genetic material.
from a plasmid to a chromosome (figure 9.27). Because trans-
posons can occur in plasmids, they can also be transmitted
from one cell to another in bacteria and a few eukaryotes. Some
transposons replicate themselves before jumping to the next
location, and others simply move without replicating first.
Prophage within
1 the bacterial
Plasmid
chromosome Transposon received from
another cell
Transposon
inserted into Chromosomal
chromosome strands
Excised phage DNA
2 contains some Regular
bacterial DNA. chromosomal
Transposon transposon
moved from site jumping to a
to site different site
or to a plasmid
New viral particles

3
are synthesized.
Transposon
removed and
spliced into
a plasmid
Plasmid with
transposon
transferred to
another cell
Infection of recipient
cell transfers bacterial
DNA to a new cell.
Figure 9.27 Transposons: shifting segments of the

genome.
5 Potential mechanisms in the movement of transposons in bacterial
cells. Some transposons are plasmids that are shifted from site to site;
others are regular parts of chromosomes that are moved out of one
Recombination results in two possible outcomes. site and spliced into another.
Process Figure 9.26 Specialized transduction:

transfer of specific genetic material by means of a virus
carrier.
genome. The smallest transposons consist of only these two
1 Specialized transduction begins with a cell that contains a
prophage (a viral genome integrated into the host cell chromosome).
genetic sequences and are often referred to as insertion ele-
2 Rarely, the virus enters a lytic cycle and, as it excises itself from its ments. A type of transposon called retrotransposon can tran-
host cell, inadvertently includes some bacterial DNA. 3 Replication scribe DNA into RNA and then back into DNA for insertion
and assembly result in production of a chimeric virus, containing in a new location. Other transposons contain additional
some bacterial DNA. 4 Release of the recombinant virus and genes that provide traits such as antibiotic resistance or toxin
subsequent infection of a new host result in transfer of bacterial DNA production.
between cells. 5 Recombination can occur between the bacterial The overall effect of transposons—to scramble the
chromosome and the virus DNA, resulting in either bacterial DNA or genetic language—can be beneficial or adverse, depending
a combination of viral and bacterial DNA being incorporated into the upon such variables as where insertion occurs in a chro-
bacterial chromosome.
mosome, what kinds of genes are relocated, and the type
of cell involved. In bacteria, transposons are known to be
involved in
Transposons contain DNA that codes for the enzymes
needed to remove and reintegrate the transposon at another 1. changes in traits such as colony morphology, pigmenta-
site in the genome. Flanking the coding region of the DNA tion, and antigenic characteristics;
are sequences called tandem repeats, which mark the point 2. replacement of damaged DNA; and
at which the transposon is removed or reinserted into the 3. the intermicrobial transfer of drug resistance (in bacteria).
■ CHECKPOINT
■ Changes in the genetic code can occur by two means: muta- ■ Mutation-induced changes in DNA nucleotide sequencing
tion and recombination. Mutation means a change in the range from a single nucleotide to addition or deletion of large
nucleotide sequence of the organism’s genome. sections of genetic material.
■ Recombination means the addition of genes from an outside ■ Genetic recombination occurs in eukaryotes through sexual
source, such as a virus or another cell. reproduction. In bacteria, recombination occurs through the
■ Mutations can be either spontaneous or induced by exposure processes of transformation, conjugation, and transduction.
to some external mutagenic agent. ■ Transposons are genes that can relocate from one part of the
■ All cells have enzymes that repair damaged DNA. When the genome to another, causing rearrangement of genetic material.
degree of damage exceeds the ability of the enzymes to make Such rearrangements have either beneficial or harmful conse-
repairs, mutations occur. quences for the organism involved.
Chapter Summary with Key Terms
9.1 Introduction to Genetics and Genes: Unlocking the (intervening sequences). The introns must be removed
Secrets of Heredity and the exons spliced together to create the final mRNA.
A. Genetics is the study of heredity and can be studied E. The genetics of viruses is quite diverse.
at the level of the organism, genome, chromosome, 1. Genomes are found in many physical forms not
gene, and DNA. Genes provide the information all of which are seen in cells, including dsDNA,
needed to construct proteins, which have structural ssDNA, and dsRNA.
or catalytic functions in the cell. 2. DNA viruses tend to replicate in the nucleus
B. DNA is a long molecule in the form of a double while RNA viruses replicate in the cytoplasm.
helix. Each strand of the helix consists of a string of Retroviruses synthesize dsDNA from ssRNA.
nucleotides that form hydrogen bonds with their
9.3 Genetic Regulation of Protein Synthesis and Metabolism
counterparts on the other strand. Adenine base-
Protein synthesis in prokaryotes can be regulated through
pairs with thymine while guanine base-pairs with
gene induction or repression, as controlled by an operon.
cytosine. The order of the nucleotides specifies which
Operons consist of several structural genes controlled by a
amino acids will be used to construct proteins during
common regulatory element.
the process of translation.
A. Inducible operons such as the lactose operon are
C. DNA replication is semiconservative and requires
normally off but can be turned on by a lactose inducer.
the participation of several enzymes.
B. Repressible operons are usually on but can be turned off
9.2 Applications of the DNA Code: Transcription and when their end product is no longer needed.
Translation C. Many antibiotics prevent bacterial growth by
A. DNA is used to produce RNA (transcription) and interfering with transcription or translation.
RNA is then used to produce protein (translation).
9.4 Mutations: Changes in the Genetic Code
Other RNAs act in regulation.
A. Permanent changes in the genome of a microorganism
B. RNA: Unlike DNA, RNA is single stranded, contains
are known as mutations. Mutations may be
uracil instead of thymine and ribose instead of
spontaneous or induced.
deoxyribose.
B. Point mutations entail a change in one or a few bases
1. Major forms of RNA found in the cell include
and are categorized as missense, nonsense, or silent
mRNA, tRNA, and rRNA.
mutations or back-mutations, based on the effect of
2. Additional regulatory RNAs include antisense
the change in nucleotide(s).
RNA, micro RNA, and ribosomids.
C. Many mutations, particularly those involving
3. The genetic information contained in DNA is
mismatched bases or damage from ultraviolet light,
copied to produce an RNA molecule. Codons
can be corrected using enzymes found in the cell.
in the mRNA pair with anticodons in the tRNA
D. The Ames test measures the mutagenicity of
to specify what amino acids to assemble on the
chemicals by determining the ability of a chemical to
ribosome during translation.
induce mutations in bacteria.
C. Transcription and Translation: Transcription occurs
when RNA polymerase copies the template strand of 9.5 DNA Recombination Events
a segment of DNA. RNA is always made in the 5’ to 3’ A. Intermicrobial transfer and genetic recombination
direction. Translation occurs when the mRNA is used permit gene sharing between bacteria. Major types of
to direct the synthesis of proteins on the ribosome. recombination include conjugation, transformation,
Codons in the mRNA pair with anticodons in the and transduction.
tRNA to assemble a string of amino acids. This occurs B. Transposons are DNA sequences that regularly move
until a stop codon is reached. to different places within the genome of a cell, as a
D. Eukaryotic Gene Expression: Eukaryotic genes are consequence generating mutations and variations in
composed of exons (expressed sequences) and introns chromosome structure.
Writing to Learn 279
Multiple-Choice and True-False Questions
Multiple-Choice Questions. Select the correct answer from the answers provided.
1. What is the smallest unit of heredity? 7. The lac operon is usually in the position and is activated
a. chromosome by a/an molecule.
b. gene a. on, repressor
c. codon b. off, inducer
d. nucleotide c. on, inducer
2. The nitrogen bases in DNA are bonded to the d. off, repressor
a. phosphate 8. Which genes can be transferred by all three methods of
b. deoxyribose intermicrobial transfer?
c. ribose a. capsule production
d. hydrogen b. toxin production
3. DNA replication is semiconservative because the strand c. F factor
will become half of the molecule. d. drug resistance
a. RNA, DNA 9. Which of the following would occur through specialized
b. template, finished transduction?
c. sense, mRNA a. acquisition of Hfr plasmid
d. codon, anticodon b. transfer of genes for toxin production
4. In DNA, adenine is the complementary base for , and c. transfer of genes for capsule formation
cytosine is the complement for . d. transfer of a plasmid with genes for degrading pesticides
a. guanine, thymine True-False Questions: If statement is true, leave as is. If it is false,
b. uracil, guanine correct it by rewriting the sentence.
c. thymine, guanine
10. The DNA pairs are held together primarily by covalent bonds.
d. thymine, uracil
11. Mutation usually has a negative outcome.
5. Transfer RNA is the molecule that
a. contributes to the structure of ribosomes 12. The lagging strand of DNA is replicated in short pieces
b. adapts the genetic code to protein structure because DNA polymerase can synthesize in only one
c. transfers the DNA code to mRNA direction.
d. provides the master code for amino acids 13. Messenger RNA is formed by translation of a gene on the
6. As a general rule, the template strand on DNA will always DNA template strand.
begin with 14. A nucleotide is composed of a 5-carbon sugar, a phosphatic
a. TAC group, and a nitrogenous base.
b. AUG
c. ATG
d. UAC
Writing to Learn
These questions are suggested as a writing-to-learn experience. For each question, compose a one- or two-paragraph answer that includes the
factual information needed to completely address the question.
1. Describe what is meant by the antiparallel arrangement 5. Compare the structure and functions of DNA and RNA.
of DNA. 6. a. Where does transcription begin?
2. On paper, replicate the following segment of DNA: b. What are the template and coding strands of DNA?
5⬘ A T C G G C T A C G T T C A C 3⬘ c. Why is only one strand transcribed, and is the same strand
3⬘ T A G C C G A T G C A A G T G 5⬘ of DNA always transcribed?
a. Show the direction of replication of the new strands and 7. The following sequence represents triplets on DNA:
explain what the lagging and leading strands are. TAC CAG ATA CAC TCC CCT GCG ACT
b. Explain how this is semiconservative replication. Are the a. Give the mRNA codons and tRNA anticodons that
new strands identical to the original segment of DNA? correspond with this sequence, and then give the sequence
3. Name several characteristics of DNA structure that enable of amino acids in the polypeptide.
it to be replicated with such great fidelity generation after b. Provide another mRNA strand that can be used to
generation. synthesize this same protein.
c. Looking at figure 9.13, give the type and order of the amino
4. Explain the following relationship: DNA formats RNA, which
acids in the peptide.
makes protein.
8. a. Summarize how bacterial and eukaryotic cells differ in 10. a. Compare conjugation, transformation, and transduction on
gene structure, transcription, and translation. the basis of general method, nature of donor, and nature of
b. Discuss the roles of exons and introns. recipient.
9. a. What is an operon? Describe the functions of regulators, b. Explain the differences between general and specialized
promoters, and operators. transduction, using drawings.
b. Compare and contrast the lac operon with a repressible
operon system.
Concept Mapping
Appendix D provides guidance for working with concept maps. 2. Construct your own concept map using the following words
as the concepts. Supply the linking words between each pair of
1. Supply your own linking words or phrases in this concept concepts.
map, and provide the missing concepts in the empty boxes.
ribozyme
Accurate copy primer
riboswitch
mRNA
Inaccurate copy
DNA tRNA
Plasmid rRNA
is transcribed into
transcription
translation
DNA
mRNA rRNA Regulatory RNAs
Protein
Critical Thinking Questions
Critical thinking is the ability to reason and solve problems using facts and concepts. These questions can be approached from a number
of angles, and in most cases, they do not have a single correct answer.
1. Knowing that retroviruses operate on the principle of 6. Explain what is meant by the expression: phenotype =
reversing the direction of transcription from RNA to DNA, genotype + environment.
propose a drug that might possibly interfere with their
replication. Try this
A simple test you can do to demonstrate the coiling of
2. Using the piece of DNA in Writing to Learn question 7,
DNA in bacteria is to open a large elastic band, stretch
show a deletion, an insertion, a substitution, and nonsense
it taut, and twist it. First it will form a loose helix, then
mutations. Which ones are frameshift mutations? Are any of
a tighter helix, and finally, to relieve stress, it will twist
your mutations nonsense? Missense? (Use the universal code
back upon itself. Further twisting will result in a series of
to determine this.)
knotlike bodies; this is how bacterial DNA is condensed.
3. Using figure 9.13 and table 9.4, go through the steps in
mutation of a codon followed by its transcription and
translation that will give the end result in silent, missense,
and nonsense mutations. Why is a change in the RNA code
alone not really a mutation?
4. Explain the principle of “wobble” and find four amino acids
that are encoded by wobble bases (figure 9.13). Suggest some
benefits of this phenomenon to microorganisms.
5. The enzymes required to carry out transcription and
translation are themselves produced through these same
processes. Speculate which may have come first in evolution—
proteins or nucleic acids—and explain your choice.
Internet Search Topics 281
Visual Understanding.
1. Figure 9.15, step 3. Label each of the parts of the 2. From chapter 4, figure 4.7. Speculate on why these cells
illustration. contain two chromosomes (shown in blue).
1
2
1
A
C
UA
P 2
E
G AC
GCU
C UG C CG
A UG
AUC
UA G
Internet Search Topics
1. Do an Internet search under the heading “DNA music.” Log on to one or more of the listed URLs and locate
Explore several websites, discovering how this music is made animations, three-dimensional graphics, and interactive
and listening to some examples. tutorials that help you to visualize replication, transcription,
2. Find information on introns. Explain at least five current and translation.
theories as to their possible functions. 4. Do an Internet search for information on Walter Benson Goad.
3. Go to: www.aris.mhhe.com, and click on “microbiology” and Where did he work? What did he help create in the 1980s, the
then this textbook’s author/title. Go to chapter 9, access the early days of molecular biology? Follow a link or do another
URLs listed under Internet Search Topics, and research the search to find information on his NIH-associated creation.The
following: Packaging of DNA: Winding, Twisting, and Coiling

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cow95289_ch09_246_281.

indd Page 246 9/17/07 6:01:23 PM admini /Volumes/207/MHIA035/cow95289indd%0/cow95289_ch09

Case File 9 Wrap-Up appears on page 249.

Organism level Cell level Chromosome level Molecular level

Figure 9.1 Levels of genetic study.

248 Chapter 9 Microbial Genetics

Chloroplast DNA RNA

CASE FILE 9 WRAP-UP

The chapter opener described the first known case of infection

The DNA Code: A Simple

250 Chapter 9 Microbial Genetics

INSIGHT 9.1 Historical

Deciphering the Structure of DNA

Sugar Nitrogen Sugar

Figure 9.4 Three views of DNA structure.

252 Chapter 9 Microbial Genetics

Figure 9.5 Simplified steps to show the semiconservative replication of DNA.

254 Chapter 9 Microbial Genetics

1 Replication origin. Short

DNA III polymerase complexes

Lagging strands Leading strands 5′

9.2 Applications of the DNA Code: Transcription and Translation 255

Their DNA is packaged in tightly wound spirals arranged

9.2 Applications of the DNA Code:

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Triplets Single nucleotide

(a) (b) Codon

Variations in the order and types

2. Proteins ultimately determine phenotype, the expression

Figure 9.8 Summary of the flow of genetic

9.2 Applications of the DNA Code: Transcription and Translation 257

INSIGHT 9.2 Discovery

Small RNAs: An Old Dog Shows Off Some New(?) Tricks

TABLE 9.2 Types of Ribonucleic Acid

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Figure 9.10 Characteristics of transfer and messenger RNA.

9.2 Applications of the DNA Code: Transcription and Translation 259

1 Overall view of a gene. RNA polymerase binding site

Process Figure 9.11 The major events in mRNA synthesis (transcription).

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these molecules and stabilizes reactions between them. The

The Master Genetic Code:

9.2 Applications of the DNA Code: Transcription and Translation 261

Second Base Position

Third Base Position

* This codon initiates translation.

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Leucine Peptide bond 2

2 Formation of peptide bond G

4 First translocation; tRNA 2 shifts into

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mRNA RNA polymerase

Figure 9.16 Speeding up the protein assembly line in bacteria.

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to be fed through more than one ribosome DNA E I E I E I E

and Translation: Similar enzymes

9.3 Genetic Regulation of Protein Synthesis and Metabolism 265