Medical Microbiology BIOL 2161

Chapter Outline
Ch. 11 – Mechanisms of Microbial Genetics

I. 11.1 The Functions of Genetic Material.

A. DNA serves two essential functions that deal with cellular information.
1. First, DNA is the genetic material responsible for inheritance and is passed from parent to offspring for
all life on earth. To preserve the integrity of this genetic information, DNA must be replicated with great
accuracy, with minimal errors that introduce changes to the DNA sequence. A genome contains the full
complement of DNA within a cell and is organized into smaller, discrete units called genes that are
arranged on chromosomes and plasmids.
2. The second function of DNA is to direct and regulate the construction of the proteins necessary to a cell
for growth and reproduction in a particular cellular environment.
B. The processes of transcription and translation are collectively referred to as gene expression. Gene expression is
the synthesis of a specific protein with a sequence of amino acids that is encoded in the gene. The flow of
genetic information from DNA to RNA to protein is described by the central dogma.
C. A cell’s genotype is the full collection of genes it contains, whereas its phenotype is the set of observable
characteristics that result from those genes. The phenotype is the product of the array of proteins being
produced by the cell at a given time, which is influenced by the cell’s genotype as well as interactions with the
cell’s environment.

II. 11.2 DNA Replication

A. Now that we know what DNA looks like, how does DNA copy itself? There were three models suggested:
1. Conservative - the parental DNA remains together, and the newly formed daughter strands are
together.
2. Semi-conservative - each of the two parental DNA strands act as a template for new DNA to be
synthesized; after replication, each double-stranded DNA includes one parental or “old” strand and one
“new” strand.
3. Dispersive - both copies of DNA have double-stranded segments of parental DNA and newly synthesized
DNA interspersed.
B. Meselson and Stahl were interested in understanding how DNA replicates.
1. They grew E. coli for several generations in a medium containing a “heavy” isotope of nitrogen ( 15N) that
gets incorporated into nitrogenous bases, and eventually into the DNA.
2. The E. coli culture was then shifted into medium containing 14N and allowed to grow for one generation.
3. DNA grown in 15N (red band) is heavier than DNA grown in 14N (orange band), and sediments to a lower
level in cesium chloride solution in an ultracentrifuge.
4. When DNA grown in 15N is switched to media containing 14N, after one round of cell division the DNA
sediments halfway between the 15N and 14N levels, indicating that it now contains 50% 14N. In
subsequent cell divisions, an increasing amount of DNA contains 14N only.
5. These results could only be explained if DNA replicates in a semi-conservative manner. Therefore, the
other two modes were ruled out.
C. During DNA replication, each of the two strands that make up the double helix serves as a template from which
new strands are copied. The new strand will be complementary to the parental or “old” strand. When two
daughter DNA copies are formed, they have the same sequence and are divided equally into the two daughter
cells. Ex: a strand of DNA with a nucleotide sequence of AGTCATGA will have a complementary strand with the
sequence TCAGTACT.
D. DNA replication in Prokaryotes. DNA replication is very complex but involves 3 main stages: initiation,
elongation, and termination
1. Initiation
a. DNA is made accessible to proteins and enzymes required for replication
b. A specific region on the DNA, called the origin, is where replication begins
c. DNA Helicase attaches here and begins to move through the DNA, unwinding it as it goes
d. This forms a Y shaped structure called replication forks.
 e. Single-strand binding proteins coat the single strands of DNA near the replication fork to
prevent the single-stranded DNA from winding back into a double helix.
2. Elongation
a. DNA polymerase (another enzyme) adds DNA nucleotides to a short primer strand using the
original DNA template strand moving in what is called the 5’ to 3’ direction. it cannot add
nucleotides if a free 3'-OH group is not available.
b. Primer – short strand of RNA that initiates DNA replication. Created by the enzyme primase.
c. A protein called the sliding clamp holds the DNA polymerase in place as it continues to add
nucleotides.
d. DNA is anti-parallel so it 5’ to 3’ ends are opposite. This results in one original strand (the
leading strand) being replicated intact while the other strand (the lagging strand) is replicated
in pieces. These pieces are called Okazaki fragments.
e. Topoisomerase prevents the over-winding of the DNA double helix ahead of the replication fork
as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then
resealing it.
3. Termination
a. DNA polymerase will continue to add nucleotides until it reaches the end of the template strand
and can progress no further
b. DNA ligase will then remove the primers, and seal the gaps between the Okazaki fragments
c. The new strand and its original complement will then rewind spontaneously
E. Summary of DNA Replication
1. DNA unwinds at the origin of replication.
2. Helicase opens up the DNA-forming replication forks; these are extended bidirectionally.
3. Single-strand binding proteins coat the DNA around the replication fork to prevent rewinding of the
DNA.
4. Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling.
5. Primase synthesizes RNA primers complementary to the DNA strand.
6. DNA polymerase starts adding nucleotides to the 3'-OH end of the primer.
7. Elongation of both the lagging and the leading strand continues.
8. RNA primers are removed by exonuclease activity.
9. Gaps are filled by DNA pol by adding dNTPs.
10. The gap between the two DNA fragments is sealed by DNA ligase, which helps in the formation of
phosphodiester bonds
F. Prokaryote vs Eukaryote
1. Eukaryotes genome is larger than prokaryotes.
2. Eukaryotes have more origins of replications, than prokaryotes
3. Eukaryotic chromosomes are linear vs the circular prokaryotes
4. The rate of replication is approximately 100 nucleotides per second, much slower than prokaryotic
replication.
5. The number of DNA polymerases in eukaryotes is much more than prokaryotes
6. The essential steps of replication are the same as in prokaryotes, but enzymes might vary.
G. Telomeres
1. At the end of a DNA strand is a six base pair sequence TTAGGG repeated 100-1000 times.
2. These sequences do not code for anything they are considered “junk DNA”
3. What they are used for is at the end of the lagging strand of DNA there is no place left for the DNA
polymerase to work so the last couple nucleotides are not paired. This causes a shortening of DNA each
time it replicates
4. Telomeres at the end allow for shortening to take place without losing necessary genes.
5. In some organisms there is an enzyme called telomerase that can replace telomeres as they are lost. For
her discovery of telomerase and its action, Elizabeth Blackburn received the Nobel Prize for Medicine
and Physiology in 2009.
6. Telomerase is only present in germ cells, adult stem cells and some cancer cells of humans and thus
shortening of telomeres is related to aging DNA repair. Most somatic cells do not make telomerase
 H. DNA Replication of Extrachromosomal Elements: Plasmids and Viruses
1. Whereas many bacterial plasmids replicate by a process similar to that used to copy the bacterial
chromosome, other plasmids, several bacteriophages, and some viruses of eukaryotes use rolling circle
replication. The circular nature of plasmids and the circularization of some viral genomes on infection
make this possible.
2. Rolling circle replication begins with the enzymatic nicking of one strand of the double-stranded circular
molecule at the double-stranded origin (dso) site.
3. In bacteria, DNA polymerase III binds to the 3’-OH group of the nicked strand and begins to
unidirectionally replicate the DNA using the un-nicked strand as a template, displacing the nicked strand
as it does so.
4. Completion of DNA replication at the site of the original nick results in full displacement of the nicked
strand, which may then recircularize into a single-stranded DNA molecule.
5. RNA primase then synthesizes a primer to initiate DNA replication at the single-stranded origin (sso) site
of the single-stranded DNA (ssDNA) molecule, resulting in a double-stranded DNA (dsDNA) molecule
identical to the other circular DNA molecule.
III. 11.3 RNA Transcription
A. Central Dogma: DNA encodes RNA, RNA encodes protein. DNA  mRNA  Protein
1. Transcription – production of mRNA from a DNA template. Transcription is relatively straightforward,
with one nucleotide being added to the mRNA strand for every nucleotide read in the DNA template
strand.
2. Translation – production of a protein from a mRNA template. Translation to protein is a bit more
complex because three mRNA nucleotides correspond to one amino acid in the polypeptide sequence
that is attached to a transfer RNA (tRNA).
3. The translation to protein is still systematic and colinear, such that nucleotides 1 to 3 correspond to
amino acid 1, nucleotides 4 to 6 correspond to amino acid 2, and so on.
B. Prokaryotes Transcription
1. Background
a. Prokaryotic DNA resides in the nucleoid, and often have abundant plasmids - shorter circular
DNA molecules that may only contain one or a few genes. Plasmids can be transferred
independently of the bacterial chromosome during cell division and often carry traits such as
antibiotic resistance.
b. Transcription in prokaryotes (and in eukaryotes) requires the DNA double helix to partially
unwind in the region of mRNA synthesis called a transcription bubble. Transcription always
proceeds from the same DNA strand for each gene - template strand. The mRNA product is
complementary to the template strand and is almost identical to the other DNA strand – non-
template strand. The only difference is that in mRNA, all of the T nucleotides are replaced with
U nucleotides.
c. The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5'
mRNA nucleotide is transcribed is called the +1 site, or the initiation site. Nucleotides preceding
the initiation site are given negative numbers and are designated upstream. Conversely,
nucleotides following the initiation site are denoted with “+” numbering and are called
downstream nucleotides.
2. Initiation.
a. Transcription begins when RNA polymerase attaches to a promoter on the DNA strand causing
the DNA helix to unwind and unzip. The polymerase comprised of all five subunits (α, α, β, β'
and σ) is called the holoenzyme.
b. The promotor is a DNA sequence onto which the transcription machinery binds and initiates
transcription. In most cases, promoters exist upstream of the genes they regulate.
c. The specific sequence of a promoter is very important because it determines whether the
corresponding gene is transcribed all of the time, some of the time, or hardly at all.
3. Elongation
a. As RNA polymerase moves along the DNA template, complimentary RNA nucleotides pair with
the DNA nucleotides of the template strand.
 b. RNA polymerase adds the RNA nucleotides in the 5’ to 3’ direction.
4. Prokaryotic Termination Signals
a. Elongation of the mRNA strand continues until RNA polymerase comes to a terminator
sequence.
b. The terminator sequence causes the RNA polymerase to stop transcribing DNA and to release
the immature mRNA transcript.
c. Depending on the gene being transcribed, there are two kinds of termination signals. One is
protein-based and the other is RNA-based.
C. Eukaryotic Transcription
1. Initiation. eukaryotes require several other proteins, called transcription factors, to first bind to the
promoter region and then help recruit the appropriate polymerase.
a. RNA polymerase I is located in the nucleolus, a specialized nuclear substructure in which
ribosomal RNA (rRNA) is transcribed, processed, and assembled into ribosomes
b. RNA polymerase II is located in the nucleus and synthesizes all protein-coding nuclear pre-
mRNAs.
c. RNA polymerase III is also located in the nucleus. This polymerase transcribes a variety of
structural RNAs that includes the 5S pre-rRNA, transfer pre-RNAs (pre-tRNAs), and small nuclear
pre-RNAs.
d. A scientist characterizing a new gene can determine which polymerase transcribes it by testing
whether the gene is expressed in the presence of a particular mushroom poison, α-amanitin.
e. Promotors. Eukaryotic promoters are much larger and more complex than prokaryotic
promoters, but both have a TATA box.
2. Elongation and Termination follow the same protocol as in prokaryotes with the polymerase
synthesizing pre-mRNA in the 5' to 3' direction.
3. Eukaryotic RNA processing.
a. The pre mRNA must be processed to mRNA before leaving the nucleus.
a. 5’ capping While the pre-mRNA is still being synthesized, a 7-methylguanosine cap is added to
the 5' end of the growing transcript by a phosphate linkage. This moiety (functional group)
protects the nascent mRNA from degradation.
b. Poly-A tail. Once elongation is complete, the pre-mRNA is cleaved by an endonuclease between
an AAUAAA consensus sequence and a GU-rich sequence, leaving the AAUAAA sequence on the
pre-mRNA. An enzyme called poly-A polymerase then adds a string of approximately 200 A
residues, called the poly-A tail.
c. Pre-mRNA splicing
i. The introns are removed by spliceosomes, a complex of enzymes that cut the primary
RNA transcript and then rejoin adjacent exons producing a mature mRNA.
ii. Introns - non-coding segments of DNA; sometimes called “junk information”.
iii. Exons - segments of DNA that will eventually be expressed as a polypeptide.

IV. 11.4 Protein Synthesis (Translation). The process of translation, or protein synthesis, the second part of gene
expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.
A. A protein sequence consists of 20 commonly occurring amino acids.
1. Each amino acid is defined within the mRNA by a triplet of nucleotides called a codon.
2. The relationship between an mRNA codon and its corresponding amino acid is called the genetic code.
3. The three-nucleotide code means that there is a total of 64 possible combinations (4 3, with four
different nucleotides possible at each of the three different positions within the codon). This number is
greater than the number of amino acids and a given amino acid is encoded by more than one codon.
This redundancy in the genetic code is called degeneracy.
4. Typically, whereas the first two positions in a codon are important for determining which amino acid will
be incorporated into a growing polypeptide, the third position, called the wobble position, is less
critical.
B. Ribosomes. Each mRNA molecule is simultaneously translated by many ribosomes, all synthesizing protein in the
same direction: reading the mRNA from 5’ to 3’ and synthesizing the polypeptide from the N terminus to the C
 terminus. The complete structure containing an mRNA with multiple associated ribosomes is called a
polyribosome (or polysome).
C. Transfer RNA (tRNA) are structural molecules and interacts with three factors: aminoacyl tRNA synthetases,
ribosomes, and mRNA.
D. Translation involves three steps:
1. Initiation
a. The reading frame is the way nucleotides in mRNA are grouped into codons. The ribosome
subunits bind to the mRNA strand in the vicinity of a start codon AUG near the 5’ end of the
mRNA.
a. The first tRNA, carrying the first amino acid (always methionine) in the protein, binds to the start
codon by matching up its anticodon. (complementary to the start codon, in this case UAC)
2. Chain Elongation
a. A second amino acid now enters the second binding site and attempts to match its anticodon to
the codon.
b. If it matches, it binds the amino acid it is carrying to the methionine of the previous tRNA by a
peptide bond.
c. The ribosome then moves down the strand causing the tRNA in the first site to shift to the
second site. This is called translocation.
d. A third amino acid now enters the newly opened second site and attempts to bind its anticodon
to the new codon. If it matches, it binds its amino acid to the previous two by the formation of a
peptide bond. Etc. Etc. Etc.
e. This process continues as long as there are appropriate codons to match with tRNA anticodons
3. Chain Termination
a. Elongation continues until the ribosome gets to a codon that signifies the end of the polypeptide
chain because no amino acid is specified.
b. This sequence is called the stop codon or nonsense codon. Stop codons are always UAA, UAG,
or UGA (Helpful phrase to remember these: U Are Annoying, U Are Gross, U Go Away)
c. The polypeptide is enzymatically cleaved from the last tRNA. The polypeptide leaves the
ribosome and then takes on its primary, secondary, tertiary and quaternary structure.
d. A good, simple 6-minute video from Professor Dave on transcription-translation
E. Protein Targeting, Folding, and Modification. During and after translation, polypeptides may need to be modified
before they are biologically active. Post-translational modifications include:
1. removal of translated signal sequences-short tails of amino acids that aid in directing a protein to a
specific cellular compartment
2. proper "folding" of the polypeptide and association of multiple polypeptide subunits, often facilitated by
chaperone proteins, into a distinct three-dimensional structure
3. proteolytic processing of an inactive polypeptide to release an active protein component, and
4. various chemical modifications (e.g., phosphorylation, methylation, or glycosylation) of individual amino
acids.

V. 11.5 Mutations. A mutation is a heritable change in the DNA sequence of an organism. The resulting organism,
called a mutant, may have a recognizable change in phenotype compared to the wild type, which is the phenotype
most commonly observed in nature. DNA polymerase can make mistakes. So, it proofreads and replaces bases as
needed.
A. Effects of Mutations on DNA Sequence. There are several types of mutations that are classified according to how
the DNA molecule is altered.
1. One type, called a point mutation, affects a single base and most commonly occurs when one base is
substituted or replaced by another.
2. Mutations can also be the result of the addition of a base, known as an insertion, or the removal of a
base, also known as deletion.
3. Sometimes a piece of DNA from one chromosome may get translocated to another chromosome or to
another region of the same chromosome; this is also known as translocation.
B. Effects of Mutations on Protein Structure and Function
 1. Some mutations are not expressed as a different amino acid; these are known as silent mutations.
2. A missense mutation results in a different amino acid being incorporated into the resulting polypeptide.
The effect of a missense mutation depends on how chemically different the new amino acid is from the
wild-type amino acid. The location of the changed amino acid within the protein also is important. For
example, if the changed amino acid is part of the enzyme’s active site, then the effect of the missense
mutation may be significant. Many missense mutations result in proteins that are still functional, at least
to some degree. Sometimes the effects of missense mutations may be only apparent under certain
environmental conditions; such missense mutations are called conditional mutations. Rarely, a
missense mutation may be beneficial.
3. A nonsense mutation converts a codon encoding an amino acid (a sense codon) into a stop codon (a
nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild
type and typically not functional.
4. Frameshift mutations caused by insertions or deletions of a number of nucleotides that are not a
multiple of three are extremely problematic because a shift in the reading frame results. Because
ribosomes read the mRNA in triplet codons, frameshift mutations can change every amino acid after the
point of the mutation. The new reading frame may also include a stop codon before the end of the
coding sequence. Consequently, proteins made from genes containing frameshift mutations are nearly
always nonfunctional.
C. Causes of Mutations.
1. Induced mutations are those that result from an exposure to chemicals, UV rays, x-rays, or some other
environmental agent.
2. Spontaneous mutations occur without any exposure to any environmental agent; they are a result of
natural reactions taking place within the body.
3. Chemical Mutagens. Various types of chemical mutagens interact directly with DNA either by acting as
nucleoside analogs or by modifying nucleotide bases.
a. Chemicals called nucleoside analogs are structurally similar to normal nucleotide bases and can
be incorporated into DNA during replication. These base analogs induce mutations because they
often have different base-pairing rules than the bases they replace. Other chemical mutagens
can modify normal DNA bases, resulting in different base-pairing rules. For example, nitrous acid
deaminates cytosine, converting it to uracil. Uracil then pairs with adenine in a subsequent
round of replication, resulting in the conversion of a GC base pair to an AT base pair. Nitrous
acid also deaminates adenine to hypoxanthine, which base pairs with cytosine instead of
thymine, resulting in the conversion of a TA base pair to a CG base pair.
b. Chemical mutagens known as intercalating agents work differently. These molecules slide
between the stacked nitrogenous bases of the DNA double helix, distorting the molecule and
creating atypical spacing between nucleotide base pairs. As a result, during DNA replication,
DNA polymerase may either skip replicating several nucleotides (creating a deletion) or insert
extra nucleotides (creating an insertion). Either outcome may lead to a frameshift mutation.
Combustion products like polycyclic aromatic hydrocarbons are particularly dangerous
intercalating agents that can lead to mutation-caused cancers. The intercalating agents ethidium
bromide and acridine orange are commonly used in the laboratory to stain DNA for visualization
and are potential mutagens.
4. Radiation. Exposure to either ionizing or nonionizing radiation can each induce mutations in DNA,
although by different mechanisms.
a. Strong ionizing radiation like X-rays and gamma rays can cause single- and double-stranded
breaks in the DNA backbone through the formation of hydroxyl radicals on radiation exposure.
Ionizing radiation can also modify bases; for example, the deamination of cytosine to uracil,
analogous to the action of nitrous acid.4 Ionizing radiation exposure is used to kill microbes to
sterilize medical devices and foods, because of its dramatic nonspecific effect in damaging DNA,
proteins, and other cellular components.
b. Nonionizing radiation, like ultraviolet light, is not energetic enough to initiate these types of
chemical changes. However, nonionizing radiation can induce dimer formation between two
adjacent pyrimidine bases, commonly two thymines, within a nucleotide strand. During thymine
 dimer formation, the two adjacent thymines become covalently linked and, if left unrepaired,
both DNA replication and transcription are stalled at this point. DNA polymerase may proceed
and replicate the dimer incorrectly, potentially leading to frameshift or point mutations.
D. DNA Repair
1. Most of the mistakes introduced during DNA replication are promptly corrected by most DNA
polymerases through a function called proofreading. In proofreading, the DNA polymerase reads the
newly added base, ensuring that it is complementary to the corresponding base in the template strand
before adding the next one. If an incorrect base has been added, the enzyme makes a cut to release the
wrong nucleotide and a new base is added.
2. If this misses one still a mismatch repair mechanism is activated. These are enzymes that recognize
mismatched bases and replace them.
3. Another type of repair is nucleotide excision repair. The DNA is unwound and separated unzipped, the
incorrect based (along with a few others on each end) are removed and replaced by DNA polymerase.
Ex: Thymine dimers
E. Identifying Bacterial Mutants. One common technique used to identify bacterial mutants is called replica
plating. This technique is used to detect nutritional mutants, called auxotrophs, which have a mutation in a
gene encoding an enzyme in the biosynthesis pathway of a specific nutrient, such as an amino acid. As a result,
whereas wild-type cells retain the ability to grow normally on a medium lacking the specific nutrient, auxotrophs
are unable to grow on such a medium. During replica plating, a population of bacterial cells is mutagenized and
then plated as individual cells on a complex nutritionally complete plate and allowed to grow into colonies. Cells
from these colonies are removed from this master plate, often using sterile velvet. This velvet, containing cells,
is then pressed in the same orientation onto plates of various media. At least one plate should also be
nutritionally complete to ensure that cells are being properly transferred between the plates. The other plates
lack specific nutrients, allowing the researcher to discover various auxotrophic mutants unable to produce
specific nutrients. Cells from the corresponding colony on the nutritionally complete plate can be used to
recover the mutant for further study.
F. The Ames Test is a method that uses bacteria for rapid, inexpensive screening of the carcinogenic potential of
new chemical compounds. The test measures the mutation rate associated with exposure to the compound,
which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk. The Ames
test uses as the test organism a strain of Salmonella typhimurium that is a histidine auxotroph, unable to
synthesize its own histidine because of a mutation in an essential gene required for its synthesis. After exposure
to a potential mutagen, these bacteria are plated onto a medium lacking histidine, and the number of mutants
regaining the ability to synthesize histidine is recorded and compared with the number of such mutants that
arise in the absence of the potential mutagen. Chemicals that are more mutagenic will bring about more
mutants with restored histidine synthesis in the Ames test. Because many chemicals are not directly mutagenic
but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at the start of
this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as
mutagenic are further tested for their potential carcinogenic properties by using other models, including animal
models like mice and rats.
VI. 11.6 How Asexual Prokaryote Achieve Genetic Diversity. Prokaryotes can share genes by three other mechanisms
using Horizontal Gene Transfer.
A. In transformation, the prokaryote takes in DNA found in its environment that is shed by other prokaryotes. If a
nonpathogenic bacterium takes up DNA for a toxin gene from a pathogen and incorporates the new DNA into its
own chromosome, it too may become pathogenic
B. In transduction, bacteriophages, the viruses that infect bacteria, sometimes also move short pieces of
chromosomal DNA from one bacterium to another. Transduction results in a recombinant organism. Archaea are
not affected by bacteriophages but instead have their own viruses that translocate genetic material from one
individual to another. Recall that in generalized transduction, any piece of chromosomal DNA may be
transferred to a new host cell by accidental packaging of chromosomal DNA into a phage head during phage
assembly. By contrast, specialized transduction results from the imprecise excision of a lysogenic prophage from
the bacterial chromosome such that it carries with it a piece of the bacterial chromosome from either side of the
phage’s integration site to a new host cell. As a result, the host may acquire new properties.
 C. In conjugation, DNA is transferred from one prokaryote to another by means of a pilus, which brings the
organisms into contact with one another. The DNA transferred can be in the form of a plasmid or as a hybrid,
containing both plasmid and chromosomal DNA. In E. coli, the genes encoding the ability to conjugate are
located on a bacterial plasmid called the F plasmid, also known as the fertility factor, and the conjugation pilus
is called the F pilus. Although typical conjugation in E. coli results in the transfer of the F-plasmid DNA only,
conjugation may also transfer chromosomal DNA. This is because the F plasmid occasionally integrates into the
bacterial chromosome through recombination between the plasmid and the chromosome, forming an Hfr cell.
D. Genetic elements called transposons (transposable elements), or “jumping genes,” are molecules of DNA that
include special inverted repeat sequences at their ends and a gene encoding the enzyme transposase.
Transposons allow the entire sequence to independently excise from one location in a DNA molecule and
integrate into the DNA elsewhere through a process called transposition. Transposons were originally
discovered in maize (corn) by American geneticist Barbara McClintock (1902–1992) in the 1940s. Transposons
have since been found in all types of organisms, both prokaryotes and eukaryotes. Thus, unlike the three
previous mechanisms discussed, transposition is not prokaryote-specific. Most transposons are nonreplicative,
meaning they move in a “cut-and-paste” fashion. Some may be replicative, however, retaining their location in
the DNA while making a copy to be inserted elsewhere (“copy and paste”). Because transposons can move
within a DNA molecule, from one DNA molecule to another, or even from one cell to another, they have the
ability to introduce genetic diversity. Movement within the same DNA molecule can alter phenotype by
inactivating or activating a gene.

VII. 11.7 Gene Regulation: Operon Theory

A. Gene expression basics
A. Proteins that are needed for a specific function, or that are involved in the same biochemical pathway,
are encoded together in blocks called operons.
B. In prokaryotic cells, there are three types of regulatory molecules that can affect the expression of
operons: repressors, activators, and inducers.
a. Repressors are proteins produced in the cell that bind to operator regions found on DNA
adjacent to the genes they control. Repressors prevent transcription of a gene in response to an
external stimulus.
b. Activators are proteins produced in the cell that bind to the promoter site found on DNA
adjacent to the genes they control. Activators increase the transcription of a gene in response to
an external stimulus.
c. Inducers are small molecules that may be produced by the cell or that are in the cell’s
environment. Inducers either activate or repress transcription depending on the needs of the
cell and the availability of substrate.
B. The trp Operon: A Repressible Operon
1. Tryptophan is an such amino acid that E. coli needs to survive by either ingestion from the environment
or synthesizing it using enzymes that are encoded by five genes. The genes are transcribed into a single
mRNA, which is then translated to produce all five enzymes.
2. If tryptophan is present in the environment, then E. coli does not need to synthesize it and the trp
operon is switched off.
3. However, when tryptophan availability is low, the switch controlling the operon is turned on, the mRNA
is transcribed, the enzyme proteins are translated, and tryptophan is synthesized.
4. The trp operon includes three important regions:
a. The coding region includes the genes for the five tryptophan biosynthesis enzymes.
b. The trp operator contains the DNA code to which the trp repressor protein can bind. However,
the repressor alone cannot bind to the operator. When tryptophan is present in the cell, two
tryptophan molecules bind to the trp repressor, which changes the shape of the repressor
protein to a form that can bind to the trp operator. Binding of the tryptophan–repressor
complex at the operator physically prevents the RNA polymerase from binding to the promoter
and transcribing the downstream genes. Because the repressor protein actively binds to the
operator to keep the genes turned off, the trp operon is said to be negatively regulated and the
proteins that bind to the operator to silence trp expression are negative regulators.
 c. The trp promoter which RNA polymerase binds to initiate transcription, is before or “upstream”
of the transcriptional start site
C. The Lac Operon: An Inducible Operon
1. Inducible operons have proteins that bind to activate or repress transcription depending on the local
environment and the needs of the cell. The lac operon encodes the genes necessary to acquire and
process the lactose from the local environment. The Z gene of the lac operon encodes beta-
galactosidase, which breaks lactose down to glucose and galactose.
2. For the lac operon to be activated, two conditions must be met.
a. The level of glucose must be very low or non-existent.
b. Lactose must be present.
3. Only when glucose is absent, and lactose is present will the lac operon be transcribed. In the absence of
glucose, the binding of the CAP protein makes transcription of the lac operon more effective. When
lactose is present, it binds to the lac repressor and changes its shape so that it cannot bind to the lac
operator to prevent transcription.
D. The Catabolite Activator Protein (CAP): A Transcriptional Activator
1. Just as the trp operon is negatively regulated by tryptophan molecules, there are proteins that bind to
the promoter sequences that act as positive regulators to turn genes on and activate them.
2. For example: when glucose is scarce, E. coli bacteria can turn to other sugar sources for fuel. When
glucose levels drop, cyclic AMP (cAMP) begins to accumulate in the cell.
3. Accumulating cAMP binds to the positive regulator catabolite activator protein (CAP), a protein that
binds to the promoters of operons which control the processing of alternative sugars. When cAMP binds
to CAP, the complex then binds to the promoter region of the genes that are needed to use the
alternate sugar sources.
4. In these operons, a CAP-binding site is located upstream of the RNA-polymerase-binding site in the
promoter. CAP binding stabilizes the binding of RNA polymerase to the promoter region and increases
transcription of the associated protein-coding genes.
E. Additional Methods of Regulation in Bacteria: Attenuation and Riboswitches
1. Attenuation whereby secondary stem-loop structures formed within the 5’ end of an mRNA being
transcribed determine if transcription to complete the synthesis of this mRNA will occur and if this
mRNA will be used for translation.
2. Riboswitch is a small region of noncoding RNA found within the 5’ end of some prokaryotic mRNA
molecules. A riboswitch may bind to a small intracellular molecule to stabilize certain secondary
structures of the mRNA molecule. The binding of the small molecule determines which stem-loop
structure forms, thus influencing the completion of mRNA synthesis and protein synthesis.
F. Other Factors Affecting Gene Expression in Prokaryotes and Eukaryotes
1. Although the focus on our discussion of transcriptional control used prokaryotic operons as examples,
eukaryotic transcriptional control is similar in many ways. As in prokaryotes, eukaryotic transcription can
be controlled through the binding of transcription factors including repressors and activators.
Interestingly, eukaryotic transcription can be influenced by the binding of proteins to regions of DNA,
called enhancers, rather far away from the gene, through DNA looping facilitated between the enhancer
and the promoter.
2. In eukaryotes, the DNA molecules or associated histones can be chemically modified in such a way as to
influence transcription; this is called epigenetic regulation. Methylation of certain cytosine nucleotides
in DNA in response to environmental factors has been shown to influence use of such DNA for
transcription, with DNA methylation commonly correlating to lowered levels of gene expression.