Download Module 7 – Microbial Molecular Biology and Genetics

NPTEL – Biotechnology – Microbiology
Module 7 – Microbial Molecular Biology and Genetics
Lecture 1 - Structure and function of genetic material
Overview:
•
Griffith's experiment showed that something caused the transformation of
bacteria.
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Avery, MacLeod, and McCarty showed that DNA was responsible, but people
were still unconvinced.
•
Hershey and Chase finally proved that DNA is the genetic material.
•
DNA is a polymer of nucleotides.
•
Chargaff’s rules
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Watson and Crick summarized the above findings and postulated their model of
DNA structure.
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A, B, Z DNA
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RNA structure
Griffith Experiment
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The bacteria in the experiment had two different strains: R strain (harmless) and S
strain (harmful).
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Live R strain had no effect on the mice, while live S strain killed the mice.
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Heat-killed S strain failed to kill the mice.
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When live R strain (harmless) was mixed with heat-killed S strain (now
harmless), and the injected mice died.
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CONCLUSION: The heat-killed cells were somehow able to retain and transfer
information.
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Fig 1. Griffith's experiment discovering a "transforming principle" in heat-killed virulent smooth pneumococcus is that it
enables the transformation of rough non-virulent pneumococcus. (This file is licensed under the Creative Commons
Attribution –Share Alike 3.0 Unported license).
Griffith experimented with two different strains of the bacteria Diplococcus
pneumoniae: strain R (rough) and strain S (smooth). The S cells have a protective
protein coat which protects them from being destroyed by the host cell's immune system.
Therefore, the R strain is harmless while the S strain is harmful. Griffith injected mice
with live strain R bacteria. The mice were found healthy and contained no living bacteria.
However, when he injected the mice with the S strain, the mice died and Griffith found
live S cells in their bodies. He then injected the mice with heat-killed S bacteria. The
mice did not die and contained no live bacteria. Nevertheless, when he injected mice with
live R cells and heat-killed S cells, the mice died. From this he concluded that the heatkilled cells, although they were not living, still passed their hereditary material to the
living R cells somehow.
Avery-MacLeod-McCarty Experiment
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Extracted components from heat-killed S bacteria.
•
After each extraction, S cells were mixed with R bacteria.
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R bacteria transformed each time until DNA was extracted from S cells.
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Avery and his colleagues concluded that DNA was the "transforming principle."
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In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty provided additional
experimental evidence using test tubes to strengthen Griffith's "transforming principle."
Like Griffith, Avery and his colleagues used harmless R bacteria to determine the genetic
factor of bacteria. First, they lysed heat-killed S cells extracted from Streptococcus
pneumonia. When the lysate combined with R bacteria, virulent S bacteria were
produced. To determine the factor responsible for transformation, Avery, MacLeod, and
McCarty removed the sugar coats, proteins, RNA, and DNA from the lysate. The R
bacteria remained non-virulent only when the DNA was removed from the lysate. In all
other cases, the R bacteria were transformed. This experiment showed that DNA was the
"transforming principle."
Hershey-Chase Experiment:
Fig 2. Hershey- Chase experimented with radioactive phosphorous and sulfur to confirm that DNA is the “Transforming
principle”. (Author: Thomasione. This is a file from the Wikimedia Commons; GNU Free Documentation License)
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Labeled bacteriophage DNA with radioactive phosphorus
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After bacteriophage infected bacteria, phosphorus was found in bacteria
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Labeled protein coat with radioactive sulfur
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After bacteriophage infected bacteria, no sulfur was found in bacteria
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Hershey and Chase provided further evidence that DNA was the "transforming
principle".
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Although Avery, MacLeod, and McCarty discovered some evidence to show that DNA
was responsible for the transfer of information, many people were still skeptical and
believed it was protein. Alfred Hershey and Martha Chase were determined to provide
more concrete evidence to prove that DNA was the genetic material in bacteriophages.
In their first experiment, Hershey and Chase labeled bacteriophage DNA by injecting
radioactive phosphorus into the bacteriophage. Because DNA contains phosphorus and
amino acids do not, only the DNA was tagged. After the bacteriophage infected a strain
of E. coli, Hershey and Chase observed radioactive phosphorus in the bacteria.
In their second experiment, Hershey and Chase injected the bacteriophage with
radioactive sulfur in order to tag only the protein coat. This time, after the bacteriophage
infected the E. coli, Hershey and Chase did not observe the presence of sulfur in the
bacteria.
From their experiments, Hershey and Chase concluded that DNA was responsible for
transferring information in Griffith’s experiment. Hershey and Chase’s experiment finally
convinced everyone of DNA’s role as the genetic material in bacteriophages.
Nucleic Acid Structure:
Nucleotides
A nucleotide is composed of a nucleobase (nitrogenous base), a five-carbon sugar
(either ribose or 2-deoxyribose), and one phosphate group. Without the phosphate group,
the nucleobase and sugar compose a nucleoside. A nucleotide can thus also be called a
nucleoside monophosphate. The phosphate group’s form bonds with the 2, 3, or 5-carbon
of the sugar, with the 5-carbon site most common. Cyclic nucleotides form when the
phosphate group is bound to two of the sugar's hydroxyl groups. Nucleotides contain
either a purine or a pyrimidine base. Ribonucleotides are nucleotides in which the sugar
is ribose. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.
Nucleic acids are polymeric macromolecules made from nucleotide monomers. In
DNA, the purine bases are adenine and guanine, while the pyrimidines are thymine and
cytosine. RNA uses uracil in place of thymine. Adenine always pairs with thymine by 2
hydrogen bonds, while guanine pairs with cytosine through 3 hydrogen bonds, each due
to their unique structures.
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Fig. 3. Nucleotide base structures: Purines and pyrimidines and its mono, di, tri phosphates.
Table 1. Naming nucleosides and nucleotides
Definitions
Adenine
(A)
Bases
Guanine
Cytosine
(G)
(C)
Uracyl
(U)
The combination of a ribose and a base
Adenosine Guanosine Cytidine
Uridine
constitutes a nucleoside.
The combination of a phosphate, a ribose
Adenylate Guanylate Cytidylate Uridylate
and a base constitutes a nucleotide.
Chargaff's rules:
Chargaff's rules state that DNA from any cell of all organisms should have a 1:1
ratio of pyrimidine and purine bases and, more specifically, that the amount of guanine is
equal to cytosine and the amount of adenine is equal to thymine. This pattern is found in
both strands of the DNA. They were discovered by Austrian chemist Erwin Chargaff.
The rule holds that a double-stranded DNA molecule globally has percentage base pair
equality: %A = %T and %G = %C. The rigorous validation of the rule constitutes the
basis of Watson-Crick pairs in the DNA double helix.
DNA as a double helix
DNA is a long polymer made from repeating units called nucleotides. As first
discovered by James D. Watson and Francis Crick, the structure of DNA of all species
comprises two helical chains each coiled round the same axis, and each with a pitch of
34 Ångströms (3.4 nanometres) and a radius of 10 Ångströms (1.0 nanometres).
According to another study, when measured in a particular solution, the DNA chain
measured 22 to 26 Ångströms wide (2.2 to 2.6 nanometres), and one nucleotide unit
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measured 3.3 Å (0.33 nm) long. Although each individual repeating unit is very small,
DNA polymers can be very large molecules containing millions of nucleotides. For
instance, the largest human chromosome, chromosome number 1, is approximately 220
million base pairs long.
Fig 4. The structure of DNA showing with detailed structure of the four bases, adenine, cytosine, guanine and thymine, and
the location of the major and minor groove.
In living organisms DNA does not usually exist as a single molecule, but instead
as a pair of molecules that are held tightly together. These two long strands entwine like
vines, in the shape of a double helix. The nucleotide repeats contain both the segment of
the backbone of the molecule, which holds the chain together, and a nucleobase, which
interacts with the other DNA strand in the helix. A nucleobase linked to a sugar is called
a nucleoside and a base linked to a sugar and one or more phosphate groups is called a
nucleotide. Polymers comprising multiple linked nucleotides (as in DNA) are called a
polynucleotide.
The backbone of the DNA strand is made from alternating phosphate and sugar
residues. The sugar in DNA is 2-deoxyribose, which is a pentose (five-carbon) sugar. The
sugars are joined together by phosphate groups that form phosphodiester bonds between
the third and fifth carbon atoms of adjacent sugar rings. These asymmetric bonds mean a
strand of DNA has a direction. In a double helix the direction of the nucleotides in one
strand is opposite to their direction in the other strand: the strands are antiparallel. The
asymmetric ends of DNA strands are called the 5′ (five prime) and 3′ (three prime) ends,
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with the 5' end having a terminal phosphate group and the 3' end a terminal hydroxyl
group. One major difference between DNA and RNA is the sugar, with the 2-deoxyribose
in DNA being replaced by the alternative pentose sugar ribose in RNA.
The DNA double helix is stabilized primarily by two forces: hydrogen bonds
between nucleotides and base-stacking interactions among the aromatic nucleobases. In
the aqueous environment of the cell, the conjugated π bonds of nucleotide bases align
perpendicular to the axis of the DNA molecule, minimizing their interaction with the
solvation shell and therefore, the Gibbs free energy. The four bases found in DNA are
adenine (abbreviated A), cytosine (C), guanine (G) and thymine (T). These four bases are
attached to the sugar/phosphate to form the complete nucleotide.
Fig. 5. Chemical structure of DNA, with colored label identifying the four bases as well as the phosphate and deoxyribose
components of the backbone.
The nucleobases are classified into two types: the purines, A and G, being fused
five- and six-membered heterocyclic compounds, and the pyrimidines, the six-membered
rings C and T. A fifth pyrimidine nucleobase, uracil (U), usually takes the place of
thymine in RNA and differs from thymine by lacking a methyl group on its ring. Uracil is
not usually found in DNA, occurring only as a breakdown product of cytosine. In
addition to RNA and DNA a large number of artificial nucleic acid analogues have also
been created to study the proprieties of nucleic acids, or for use in biotechnology.
Twin helical strands form the DNA backbone. Another double helix may be
found by tracing the spaces, or grooves, between the strands. Grooves are adjacent to the
base pairs and may provide a binding site. As the strands are not directly opposite each
other, the grooves are unequally sized. One groove, the major groove, is 22 Å wide and
the other, the minor groove, is 12 Å wide. The narrowness of the minor groove means
that the edges of the bases are more accessible in the major groove. As a result, proteins
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like transcription factors that can bind to specific sequences in double-stranded DNA
usually make contacts to the sides of the bases exposed in the major groove.
In a DNA double helix, each type of nucleobase on one strand normally interacts
with just one type of nucleobase on the other strand. This is called complementary base
pairing. Here, purines form hydrogen bonds to pyrimidines, with A bonding only to T,
and C bonding only to G. This arrangement of two nucleotides binding together across
the double helix is called a base pair. As hydrogen bonds are not covalent, they can be
broken and rejoined relatively easily. The two strands of DNA in a double helix can
therefore be pulled apart like a zipper, either by a mechanical force or high temperature.
As a result of this complementarity, all the information in the double-stranded sequence
of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed,
this reversible and specific interaction between complementary base pairs is critical for
all the functions of DNA in living organisms.
The two types of base pairs form different numbers of hydrogen bonds, AT
forming two hydrogen bonds, and GC forming three hydrogen bonds. DNA with high
GC-content is more stable than DNA with low GC-content.
As noted above, most DNA molecules are actually two polymer strands, bound
together in a helical fashion by noncovalent bonds; this double stranded structure
(dsDNA) is maintained largely by the intrastrand base stacking interactions, which are
strongest for G, C stacks. The two strands can come apart – a process known as melting –
to form two ss DNA molecules. Melting occurs when conditions favor ssDNA; such
conditions are high temperature, low salt and high pH (low pH also melts DNA, but since
DNA is unstable due to acid depurination, low pH is rarely used).
A, B and Z DNA
In a DNA molecule, the two strands are not parallel, but intertwined with each
other. Each strand looks like a helix. The two strands form a "double helix"
structure, which was first discovered by James D. Watson and Francis Crick in 1953. In
this structure, also known as the B form, the helix makes a turn every 3.4 nm, and the
distance between two neighboring base pairs is 0.34 nm. Hence, there are about 10 pairs
per turn. The intertwined strands make two grooves of different widths, referred to as the
major groove and the minor groove, which may facilitate binding with specific proteins.
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In a solution with higher salt concentrations or with alcohol added, the DNA
structure may change to an A form, which is still right-handed, but every 2.3 nm makes a
turn and there are 11 base pairs per turn.
Another DNA structure is called the Z form, because its bases seem to zigzag. Z
DNA is left-handed. One turn spans 4.6 nm, comprising 12 base pairs. The DNA
molecule with alternating G-C sequences in alcohol or high salt solution tends to have
such structure.
RNA
Each nucleotide in RNA contains a ribose sugar, with carbons numbered 1'
through 5'. A base is attached to the 1' position, in general, adenine (A), cytosine (C),
guanine (G), or uracil (U). Adenine and guanine are purines, cytosine, and uracil are
pyrimidines. A phosphate group is attached to the 3' position of one ribose and the 5'
position of the next. The phosphate groups have a negative charge each at physiological
pH, making RNA a charged molecule (polyanion).
An important structural feature of RNA that distinguishes it from DNA is the presence of
a hydroxyl group at the 2' position of the ribose sugar. The presence of this functional
group causes the helix to adopt the A-form geometry rather than the B-form most
commonly observed in DNA. This results in a very deep and narrow major groove and a
shallow and wide minor groove. A second consequence of the presence of the 2'hydroxyl group is that in conformationally flexible regions of an RNA molecule (that is,
not involved in formation of a double helix), it can chemically attack the adjacent
phosphodiester bond to cleave the backbone.
RNA is transcribed with only four bases (adenine, cytosine, guanine and uracil), but these
bases and attached sugars can be modified in numerous ways as the RNAs mature.
Pseudouridine (Ψ), in which the linkage between uracil and ribose is changed from a C–
N bond to a C–C bond, and ribothymidine (T) are found in various places (the most
notable ones being in the TΨC loop of tRNA). Another notable modified base is
hypoxanthine, a deaminated adenine base whose nucleoside is called inosine (I). Inosine
plays a key role in the wobble hypothesis of the genetic code.
There are nearly 100 other naturally occurring modified nucleosides, of which
pseudouridine and nucleosides with 2'-O-methylribose are the most common. The
specific roles of many of these modifications in RNA are not fully understood.
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The functional form of single-stranded RNA molecule requires a specific tertiary
structure. The scaffold for this structure is provided by secondary structural elements that
are hydrogen bonds within the molecule. This leads to several recognizable "domains" of
secondary structure like hairpin loops, bulges, and internal loops. Since RNA is charged,
metal ions such as Mg2+ are needed to stabilize many secondary and tertiary structures.
REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
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Module – 7 Microbial Molecular Biology and Genetics
Lecture 2 - DNA replication
DNA replication
Templated replication
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Sense and antisense strands: The sense strand of a double-stranded DNA
molecule has a base sequence similar to that of the RNA that is transcribed from
the DNA. The antisense strand (also known as template strand) has a sequence
that is the reverse complement of the sense strand (base paired to it with an
antiparallel 5' to 3' polarity).
•
Semiconservative replication: Discovery of the double helical structure of DNA
immediately suggested a mechanism for precise replication -- namely separation
of the two strands and template synthesis of a new copy of each of the missing
complementary strands. This model predicted that each new double helix should
have one strand of parental DNA and one strand of newly synthesized DNA.
Fig 6. Three postulated methods of replication: 1. Semiconservative, 2. Conservative, 3. Dispersive
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•
Messelson-Stahl experiment: Semiconservative replication was demonstrated by
starting with bacteria whose DNA was labeled with heavy nitrogen (15N) and then
allowing various amounts of growth to occur in media labeled with normal
nitrogen. After one round of replication, the entire DNA had an intermediate
density. After two rounds, one half was light and the other half still had an
intermediate density. There was no conservation of heavy DNA. However, the
data suggested that each of the labeled strands from the original DNA remained
intact and separated from its partner in each replication cycle.
Initiation of replication
•
Origin of Replication: The DNA of a bacterial chromosome is a closed circular
structure. DNA replication begins at a specific site (origin of replication)
characterized by the presence of repeated 9 base and 13 base nucleotide
sequences. The repeating units are referred to as 9-mers and 13-mers. The 13
mers are AT rich, making easier to separate the two strands of the double-stranded
DNA.
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DNAa protein: The protein coded by the DNA a gene binds to the repeated
9mers. This forms a tight loop and generates a strain that causes strand separation
in the region containing the AT-rich 13-mers
•
Helicase: Enzymes called helicases use energy derived from ATP to further
separate the two strands of the DNA double helix.
•
Topoisomerase I: Separation of the two strands of the DNA double helix requires
substantial unwinding of the helix. An enzyme known as topoisomerase I (gyrase)
relieves twisting strain that is generated by unwinding the double helix. It is
believed to act by cutting one of the strands such that the other strand can rotate
freely to relieve the strain, and then resealing the strand that has been temporarily
cut.
•
Keeping the helix open: Single stranded DNA-binding proteins (SSBPs) attach
to the single stranded DNA generated by unwinding the double helix and
temporarily keep it from reforming double helical structures.
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Patterns of replication
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Replication forks: When the two strands of double helical DNA separate and
replication of both strands begins, a forked or Y-shaped structure is formed.
•
Bidirectional replication: In bacterial cells, replication starts at a specific origin
of replication within the circular DNA molecule and proceeds in both directions
away from the origin. This results in the formation of a replication "bubble",
which continues to elongate as replication proceeds. A similar pattern is seen in
eukaryotic chromosomes, except that multiple origins are involved.
•
Theta structure in bacteria: As replication proceeds in both directions around
the circular chromosomes of bacteria, a structure reminiscent of the Greek letter
theta is formed.
•
Replicons: The very long DNA double helices found in eukaryotic chromosomes
contain multiple origins of replication, which often initiate bidirectional
replication more or less synchronously. Each unit of replication is called a
replicon. Replication continues until the replication bubbles fuse to yield fully
replicated DNA strands.
•
Other patterns There are also two alternative strategies for replication of circular
DNA that are more complex than the simple bidirectional model. These are the
rolling circle mode (sigma mode) used by some types of viruses, and the D loop
mode, used for replication of mitochondrial and chloroplast DNAs.
5'-to-3' synthesis
•
Unidirectional addition of nucleotides: Polymerization of DNA, and also of
RNA, occurs by condensing a 5'-nucleoside triphosphate (dNTP or NTP) onto the
3' hydroxyl group of another nucleotide, or onto the 3'-end of a growing
polynucleotide chain.
No mechanism exists for extending the 5'-end of a
nucleotide chain.
•
Energy for synthesis: Hydrolysis of the dNTP or NTP that is being added
provides the energy needed to form a covalent bond. The outer two phosphates of
the triphosphate are split off and the innermost phosphate forms an ester linkage
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with the 3'-hydroxyl group at the end of the pre-existing chain. This results in a
phosphodiester bond
RNA priming
•
No new DNA starts: Deoxynucleotides can only be added to 3'-end of a preexisting strand of DNA (or RNA). There are no enzymes capable of initiating the
synthesis of a DNA-templated DNA molecule at the level of a single nucleotide.
This makes necessary to use an indirect priming procedure.
•
RNA primers: A variety of enzymes are capable of initiating new DNAtemplated RNA synthesis (as in transcription). New DNA synthesis is primed
with a short segment of RNA that is later removed
•
Primase: A separate enzyme in the initiation complex called primase synthesizes
a short RNA primer each time that new DNA synthesis begins, including all new
starts in the discontinuous pattern of synthesis described below.
Leading and lagging strands
•
Unidirectional synthesis of antiparallel DNA: The inability to synthesize new
chains in a 3' to 5' direction adds a major complication to the replication of
antiparallel double-stranded DNA. At any replication fork, one of the template
strands has a 3' to 5' orientation, which is what is needed for synthesis of a new
complementary strand in a 5' to 3' direction. Synthesis on that template is primed
and starts very quickly. However, the other template strand has a 5' to 3'
orientation and is thus unable to support synthesis beginning at the origin and
moving away from it in a 3' to 5' direction. Because of this, the 5' to 3' template
strand accumulates in a single stranded configuration until there is a sufficient
length so that synthesis of its complementary antiparallel strand can be primed
and initiated in a 5'-to-3' direction ("backward" toward the origin of replication).
The strand whose synthesis begins immediately is called the "leading" strand, and
the one whose synthesis is delayed is called the "lagging" strand.
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•
Discontinuous synthesis -- Okazaki fragments: As synthesis of the leading
strand continues, more and more of the single stranded DNA of the lagging strand
is unwound. Each time that a sufficient length is reached, synthesis of a new
segment of the complementary strand begins. If the replicating DNA is denatured
(separated into individual strands) before the newly synthesized pieces of the
lagging strand have been ligated together, a number of relatively small fragments
of newly synthesized DNA will be recovered, together with the much longer
strands produced by continuous synthesis in the leading strand. The small
fragments are called "Okazaki fragments", named for the person who first
discovered them.
DNA polymerase III
Template DNA synthesis: After the RNA primer has reached an adequate length,
DNA polymerase III begins synthesis of DNA, which proceeds to completion in
the leading strand, and proceeds until the 5'-end of the previous primer is
encountered in the lagging strand.
•
DNA polymerase III holoenzyme: DNA polymerase III is a highly complex
dimeric aggregate, consisting of 20 or more protein subunits. The alpha subunits
perform the actual DNA synthesis, but operate in conjunction with multiple
accessory proteins
•
Simultaneous synthesis of leading and lagging strands: There is yet another
complicating factor in DNA synthesis. It is now generally believed that the
leading and lagging strands are synthesized simultaneously by a single dimeric
DNA polymerase III complex. This requires formation of a looped structure with
the leading and lagging strands so positioned that their synthesis can occur side by
side in the same orientation, despite the fact that the newly synthesized chains are
growing in opposite directions relative to the overall DNA that is being replicated.
A similar looping also appears to occur in eukaryotic DNA replication.
•
Clamping function of beta subunits: The beta subunits appear to have a
clamping function that keeps the leading and lagging strands appropriately
aligned with the catalytically active alpha subunits.
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•
Replisome complex: The entire DNA-synthesizing complex at each replication
fork, which also includes topoisomerase, helicase, and primase, is sometimes
referred to as a replisome.
DNA polymerase I
•
Not the primary enzyme for DNA synthesis: DNA polymerase I is so named
because it was the first of the DNA polymerase enzymes to be isolated and
characterized. Although it is capable of template-directed DNA synthesis, it is
now known not to be the enzyme primarily responsible for new DNA synthesis,
but it does have other important roles, as described below.
•
5' to 3' exonuclease activity: DNA polymerase I has an unusual 5' to 3'
exonuclease activity. This gives it the ability to start at a single-stranded break
and progressively remove nucleotides and replace them in a 5' to 3' direction.
•
Removal of RNA primer: In the lagging strand, when DNA polymerase III runs
into the 5'-end of the previous RNA primer, it is unable to proceed further.
Although the new DNA butts up against the primer, it is not covalently joined to
it. DNA polymerase III dissociates, leaving a "nick" (a single stranded gap)
between the new DNA and the primer. Starting at the nick, DNA polymerase I
removes the primer ribonucleotides one at a time, using its 5' to 3' exonuclease
activity, and replaces them with deoxyribonucleotides, using its DNA polymerase
activity..
•
DNA repair: DNA polymerase I is also used to fill in short gaps in DNA, often
as a part of a repair process that excises part of a damaged strand and replaces it
with new DNA templated from the remaining strand.
Proofreading and DNA repair
•
Removal of mismatched bases: DNA polymerases III and I both have 3' to 5'
exonuclease activity. This allows them to remove a mismatched nucleotide that
has just been added to a growing DNA chain and make another attempt to insert
the correct nucleotide. This "proofreading" function helps to reduce the number of
mistakes in DNA synthesis that would otherwise result in mutation..
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•
DNA polymerase II: There is yet another prokaryotic DNA polymerase,
designated DNA polymerase II, whose main function appears to be to synthesize
replacement DNA during DNA repair.
Final steps in DNA synthesis
•
DNA Ligase: After the last ribonucleotide is removed from the primer and
replaced with a deoxynucleotide, there is still a nick in the newly synthesized
lagging strand. This nick is closed by DNA ligase, which forms a covalent
phosphodiester bond between the Okazaki fragments, joining them into a
continuous strand of DNA. Note that Okazaki fragments accumulate when ligase
function is impaired.
•
Topoisomerase II: Its role is to cut and reseal the newly synthesized DNAs as
needed so that they can separate from each other (it is easy to visualize two
circular genomes linked through each other at the end of replication).
Fig. 7. Diagram of DNA polymerase extending a DNA strand and proof-reading.
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REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
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Module – 7 Microbial Molecular Biology and Genetics
Lecture 3 - The genetic code and the gene structure
The Genetic code
Since there are 20 different kinds of amino acids in proteins and only four kinds
of nucleotides in DNA, the relationship between the gene and its most elementary
functional product, i.e., between DNA and protein, can hardly be interpreted through a
code of one nucleotide = one amino acid. A coding sequence of two nucleotides for one
amino acid, or a doublet code, would produce only 16 possible coding combinations, or
codons. Codons are group of nucleotides that specifies one amino acid. By the genetic
code (George Gamov, 1954), we mean, a collection of base sequences (codons) that
correspond to each amino acid and to translation signals. A codon size of three
nucleotides for one amino acid are triplet codon seems more likely, since it produces 64
possible codons, however, only 20 amino acids need to be coded, 44 codons in a triplet
code seem to be superfluous. To account for the excess of codons beyond the necessary
20, we can suppose that more than one codon can code for a particular amino acid. For
example, if each kind of amino acid were coded by three different possible codons, 60
possible codons would be accounted for. A code in which there is more than one codon
for the same amino acid, is called degenerate. It is also possible that some or all of the
codons in excess of 20 do not code for any amino acid and are therefore nonsense
codons.
Fig. 8. Genetic Code.
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The genetic code has following general properties, mostly applicable to the genes of all
the organisms:
Genetic code is triplet. As discussed earlier, singlet and doublet codons cannot form 20
combinations, which is the minimum requirement, therefore triplet codon is a necessity,
so that all the amino acids must be coded.
Genetic code is non-overlapping. During translation, the codons are read one after
another, in a sequence. One base of a codon is not used by the other codons.
Therefore, if there are six bases, they will code for two amino acids only. e.g.; in case
of non-overlapping, a gene sequence of UUUCCC only two amino acids will be
coded, phenylalanine (UUU) and proline (CCC), whereas for an overlapping code,
more than two amino acids could be coded, phenylalanine (UUU), serine (UCC) and
proline (CCC).
Fig. 9. Overlapping and non-overlapping genetic code
Genetic code is commaless. The bases are read one after the other in the codons, i.e., no
bases or codons are reserved for punctuation or comma. When the first amino acid is
coded, the second will be coded by the next three bases immediately, and no base will be
wasted to serve as a comma. Once the translation begins, the codons are read one after
the other with no break or demarcating signals in between them.
Genetic code is non-ambiguous. Each codon has a particular amino acid for coding, and
it will code for that amino acid only. There is one to one relationship between codon to
amino acid. However, there is an exception, AUG codes for methionine and GUG codes
for valine, but if AUG is absent, then GUG codes for methionine, as starting codon for
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protein synthesis. In an ambiguous code, one codon can code for more than one amino
acid.
In certain rare cases, the genetic code is found to be ambiguous i.e, some codons codes
for different amino acids under different conditions, e.g. in streptomycin sensitive strain
of E.coli the codon UUU normally codes for phenylalanine, but it may also code for
isoleucine, serine when treated with streptomycin. This ambiguity is enhanced at high Mg
ion concentration, low temperature and the presence of ethyl alcohol.
Genetic code is degenerate. Since there are more codons than the amino acids, more
than one codon may specify the same amino acid. Such different codons that specify the
same amino acid are called as synonymous codons. e.g., UUU = UUC = phenylalanine.
Genetic code has start/stop signals. Some codons are specially meant for initiation and
termination of protein synthesis. e.g.: AUG codes for methionine, serves as initiation
codon in eukaryotes and GUG in case of prokaryotes. Three codons UAG (amber), UAA
(ochre), UGA (opal) are called as termination codons, because they terminate protein
synthesis. Earlier, they were called as nonsense codons, because they do not code for any
amino acid, but, since they are involved in termination of protein synthesis, they are
called as termination codons. The initiation and termination codons are known as signals
and this phenomenon is known as punctuation.
Genetic code is polar. It means that the genetic code has a fixed start and termination
ends, and is always read in a fixed direction, i.e. in 5’→ 3’ direction and the polypeptide
chain is synthesized in N→C direction i.e., from amino group (NH2) to carboxylic group
(COOH).
Genetic code is universal. The same genetic code is applicable to all organisms, from
bacteria to man, i.e.; the codons have the same meaning in all the organisms. e.g., UUU =
phenylalanine in bacteria, mouse, man and tobacco. The universality of the genetic code,
however, does not mean that DNA base ratios must be similar in different species for
genes specifying similar proteins. The fact that the code is degenerate enables many bases
to be changed by mutation in a sequence of mRNA, but this mRNA could still produce
the same amino acid sequence.
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In 1979 investigators began started DNA sequencing of mitochondrial DNA in
humans, cattle and mice. During their experiments, they were surprised to learn that the
genetic code used by the mitochondrial DNA was not the same as the universal genetic
code. e.g. UGA, which is a non-sense codon, but it codes for tryptophan in mtDNA,
AGG which codes for arginine is a non-sense codon in mtDNA. So, extra chromosomal
DNA such as mtDNA and ctDNA do not come under the universality of the genetic code.
Wobble Hypothesis. Out of the 64 codons, three are involved in termination process. So,
there are only 61 codons specifying the amino acids, and the cell should have 61 different
types of tRNAs, each having a different anticodon for the recognition of codons.
However, the actual number of tRNA is found to be much less than 61. This means that
the anticodons of tRNA read more than one codon on the mRNA. Crick (1966) proposed
a hypothesis to explain the degeneracy of the genetic code; the hypothesis is known as
Wobble hypothesis.
According to this hypothesis, the major degeneracy occurs at the third position, i.e., the
third codon is not important in base pairing, and the actual pairing occurs only in the first
two codon-anticodon pairs. The base at the 5′ end of the anticodon and the base at the 3′
end of the codon form hydrogen bonds without any specificity. The third base is called as
the wobble base. This wobble base of codon lacks specificity and the base in the first
position of the anticodon is usually abnormal e.g., inosine, tyrosine, etc. These abnormal
bases are able to pair up with more than one nitrogen base at the same position e.g.,
inosine (I) can pair up with A, C and U. The pairing between unusual base of tRNA and
wobble base of mRNA is called wobble pairing.
Gene structure:
A gene is a linear sequence of nucleotides or codons (= follow-up of three
nucleotides)
with a fixed start and end point. In its most simplified version one gene codes the
information for one enzyme (--> "one gene - one enzyme hypothesis"). This hypothesis
has been modified over the years to the "one gene - one polypeptide" hypothesis, which
holds true for most bacterial genes. Many enzymes and proteins for which genes carry the
code
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are made up from more than one polypeptide requiring more than one gene. "The DNA
segment which codes for a single polypeptide sequence is often referred to as a cistron."
Most genes consist of discrete sequences of codons that are "read" (transcribed)
on only one way to generate one translated polypeptide sequence. The code is NOT
overlapping and there is only one defined start point with one reading frame, i.e. the way
in which the DNA nucleotides are arranged in codons. While each gene harbors the
genetic information of the DNA strand and typically codes for a specific protein or
enzyme, viral, prokaryotic and eukaryotic genes structures differ significantly. In
contrary to bacterial genes, genes in eukaryotic life forms are more spaced apart and also
are organized in so-called exon-intron structure;
- exons are coding segments of a
genes, while introns are non-coding gene regions which play a role in splicing and gene
regulation; - genetic information of cistrons in prokaryotes is usually continuous and
introns rarely occur; moreover, bacterial genes are often organized in so-called operons;
- transcription of bacterial operons generates polygenic or polycistronic mRNA, i.e.
which is RNA that is translated into more than one polypeptide.
Fig. 10. A bacterial structural gene.
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Functional structure of a gene:
Fig 11. Diagram of the "typical" eukaryotic protein-coding gene. Promoters andenhancers determine what portions of
the DNA will be transcribed into theprecursor mRNA (pre-mRNA). The pre-mRNA is then spliced into messenger
RNA(mRNA) which is later translated into protein.
•
All genes have regulatory regions in addition to regions that explicitly code for a
protein or RNA product. A regulatory region shared by almost all genes is known
as the promoter, which provides a position that is recognized by the transcription
machinery when a gene is about to be transcribed and expressed. A gene can have
more than one promoter, resulting in RNAs that differ in how far they extend in
the 5' end. Although promoter regions have a consensus sequence that is the most
common sequence at this position, some genes have "strong" promoters that bind
the transcription machinery well, and others have "weak" promoters that bind
poorly. These weak promoters usually permit a lower rate of transcription than the
strong promoters, because the transcription machinery binds to them and initiates
transcription
less
frequently.
Other
possible
regulatory
regions
include enhancers, which can compensate for a weak promoter. Most regulatory
regions are "upstream"—that is, before or toward the 5' end of the transcription
initiation site. Eukaryotic promoter regions are much more complex and difficult
to identify than prokaryotic promoters.
•
Many prokaryotic genes are organized into operons, or groups of genes whose
products have related functions and which are transcribed as a unit. By
contrast, eukaryotic genes are transcribed only one at a time, but may include long
stretches of DNA called introns which are transcribed but never translated into
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protein (they are spliced out before translation). Splicing can also occur in
prokaryotic genes, but is less common than in eukaryotes.
Of the two nucleotide strands of the DNA of a gene, only one strand contains
coded genetic information and directs synthesis of RNA via a cell process called DNA
transcription. This strand is called the template strand. During DNA transcription, only
one strand of the double-helix of a gene, the so-called sense or template strand, serves as
the so-called template for DNA transcription to form the new RNA molecule. While
during DNA replication certain enzymes called helicases help to unwind and open the
DNA double helix at the ori, it is the RNA polymerase enzyme which unwinds DNA
during DNA transcription to create single stranded DNA regions.The distinct places
along the DNA strand where transcription begins are called transcription start sites. - at
the transcription start site, a complex protein cluster, the so-called transcriptome,
assembles itself; in eukaryotic cells, the transcriptome is
comprised of many protein
components. Along these unwind and single-stranded DNA regions, new nucleotides are
paired according to the Watson-Crick base-pairing rule (--> A with T; and G with C).
The polymerization of the new nucleotides during the process of DNA transcription
is catalyzed by an enzyme called RNA polymerase (RNA POL). The RNA polymerase
recognizes certain structures on the DNA strand, called promoter regions, or promoters.
Promoters are sequences of DNA which are usually located upstream from the actual
coding or transcribed region of a gene. Different genes have promoters with different
DNA sequences. In E.coli the promoter has two different functions which relate to two
different regions within the promoter, each with a unique consensus sequence:
1. RNA polymerase recognition site
- Mediated by the RNA polymerase recognition site, with the consensus sequence
5'-TTGACA-3' on the non-template strand of the gene
- The site of the initial association of the RNA polymerase enzyme with the gene
- Located about 35bp (-35 region) away from the transcription start site
2. RNA polymerase binding site
- this DNA sequence is also known as the "Pribnow box"
- centered at the -10bp region of the gene
- this is the region where the RNA polymerase enzyme starts to localized unwinding of
the
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DNA double strand of the gene
- both gene regions tells the ribosome where to dock on and where to start the DNA
transcription process on a gene
- promoter regions often contain important regulatory sites, such as the operator region or
the CAP binding site (see bacterial gene regulation!), which allow the fine tuning of
DNA transcription depending on different environmental conditions.
Another important stretch of DNA on a gene is the so-called leader sequence.
- this gene region located downstream of the promoter region is transcribed first into
mRNA, but is does NOT contain a translated codon;
- it is a non-translated DNA sequence with two known functions:
1. it is important for initiation of protein translation at the small subunit of the
ribosome
- this happens with the help of the Shine-Dalgarno consensus sequence, (5'-AGGA3'),
which is complementary to a sequence on the 16S rRNA transcript
2. it is involved in the regulation of DNA transcription, e.g. during attenuation.
Following the non-transcribed leader sequence of a gene follows the coding region.
- this code carrying region directs the actual synthesis of the polypeptide chain at the
ribosome
- a coding sequence of a gene usually begins with the consensus sequence 5'-TAC-5';
- this sequence after transcription by the RNA polymerase into the RNA translation
initiation codon 5'-AUG-3' is translated by the ribosome into the amino acid Nformylmethionine;
- the remainder of the coding region of a gene consists of a unique sequence of triplet
codons, e.g. UCG, that specifies the sequence of amino acids for that particular gene;
Another important sequence or region on a typical gene is the terminator sequence.
- these gene region located at the end of a gene sequence is important to enforce the
termination of DNA transcription by stopping the RNA polymerase;
- it often after a non-transcribed so-called trailer sequence, which is important for the
proper
expression of the coding region of a gene.
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In order to begin gene transcription, the RNA polymerase enzyme further requires a
number of helper proteins, the so-called transcription factors (TFs)
- important bacterial transcription factors Sigma and Rho;
- important eukaryotic transcription factors are myc, AP-1, TFIIA, TFIIB, etc.;
- the RNA polymerase plus the transcription factors recognize and bind to the so-called
promoter region (TATA box), which is located shortly before the beginning of a typical
gene
REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
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Module 7 – Microbial Molecular Biology and Genetics
Lecture 4 - The expression of genes
Expression of genes
•
DNA serves two major functions:
1. Encoding structural information that can be converted into RNA and
(usually), hence into protein sequence.
2. Encoding regulatory signals that allow certain proteins to decide where
to begin or terminate reading DNA
•
The "central dogma" of molecular biology: information flows from nucleic acids
to proteins, not the reverse:
DNA ----> RNA ----> polypeptides (proteins)
Transcription = the process of making RNA from DNA templates
•
Translation = the process of making polypeptides
Transcription
•
Transcription is a fundamental cellular process: RNA polymerases "transcribe"
the genetic information on DNA into RNA strands. All cells have RNA
polymerases (RNAP).
•
The RNA polymerases increase in complexity as you go from viruses (example,
T7 RNA polymerase is made up of a single protein), to bacterial systems (one
RNA polymerase made up of the proteins - beta, beta', 2 x alpha, omega and the
sigma factor), and finally to eukaryotic systems (Three RNA polymerases - Pol I,
Pol II, and Pol III, each with ten or more subunits).
•
While the RNA polymerases have become increasing complex as life evolved,
their overall structure (as evidenced by crystallographic structures of bacterial
RNA polymerase and Pol II) show remarkable similarity. There is also sequence
similarity between the bacterial polymerase protein subunits and the proteins that
make up the eukaryotic polymerases.
•
Transcription of any gene usually involves three distinct stages:
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o
First, the RNA polymerase has to find the start site of a gene. The
"holoenzyme" form of the polymerase does this by looking for the
"promoter" site that exists just upstream of the gene start site. This process
is termed "transcription initiation". This is followed by opening up
(melting) of the duplex DNA to form an "open complex".
o
This is followed by a rapid change into the "elongation" phase of
transcription where the "core polymerase" part of the RNA polymerase
rapidly transcribes an RNA strand that is complementary to the "template"
strand of the DNA. The change into the elongation phase usually occurs
after a few bases of RNA have been transcribed (typically about 8-9 bases
of RNA in bacterial system which form a RNA-DNA hybrid with the
template strand), and involves a "clamping down" on the DNA to prevent
the polymerase from falling off the DNA.
o
The final stage of the transcription of a gene is "termination", after the
stop codon of the gene. The process of termination usually involves
sequences where the polymerase slows down or stalls, and the
polymerase-RNA-DNA complex (often proteins such as rho and NusA are
involved in bacterial systems).
•
Genes have to be transcribed to mRNAs before they can be translated into
proteins; more or less mRNA from a particular gene equals more or less of the
protein encoded by the gene. Transcription is, thus, an important point in the
control of gene "expression". Most genes are controlled transcriptionally, usually
by regulation of the level of transcriptional initiation. For example, if a gene has a
strong promoter, it will be more highly expressed when compared to another gene
with a weak promoter site. Similarly if a regulatory protein can bind the promoter
site of a gene (and prevent transcription initiation), then it can turn off the
expression of that gene.
•
Transcriptional control of genetic expression is vital for cellular functions, and
many diseases and cancers are results of defects in the transcriptional control of
essentials genes.
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Transcription initiation
•
Transcription initiation in bacteria (prokaryotes) involves sigma factors. The sigma
factor combine with the core RNA polymerase to form a holoenzyme that is
competent for promoter binding. The core RNA polymerase, by itself, cannot bind the
promoter site.
•
The sigma factor can be thought of as the specificity factor in the RNA polymerase.
Each bacteria has several different sigma factors that recognize slightly different
promoter sequences. Predominant among these is the sigma70 (70 kDa in E. coli; also
called rpoD), which initiates the transcription of most genes in exponentially growing
cells. There are two general classes of sigma factors - sigma 70 class and sigma 54
class:
•
The sigma 70 class of sigma factors share extensive sequence homology, and bind to
two conserved sequences upstream of the gene start site (the -35 box and the -10
box). Each of these sigma factors recognizes slight variations in these conserved
sequence boxes. Sigma70 binds promoter DNA as a part of the holoenzyme, and
binds DNA very poorly in absence of the core enzyme.
•
The sigma54 class of genes (54 kDa in E. coli; also called rpoN) controls a much
smaller set of genes than sigma70. It recognizes different conserved sequences (the 12 and -24 boxes). Unlike Sigma70, sigma54 can bind DNA even in the absence of
the core RNA polymerase. It however lacks the ability to melt promoter DNA on its
own - for this it needs to interact with other activator proteins that bind further
upstream of the promoter site, as well as the core RNAP.
•
In eukaryotic systems, transcription initiation is very different. There are no sigma
factors. Instead, the central protein in forming the "pre-initiation complex " (PIC) is
the TATA binding protein (TBP), that binds to the TATA box, a conserved sequence
just upstream of the initiation region. A large number of other general transcription
factors such as TFIIB, TFIIE, TFIIF, TFIIH (TF stands for transcription factor; II
stands for Pol II; there are similar factors for Pol I and Pol II) and others assemble to
form the multisubunit TFIID complex. This PIC then recruits the RNA polymerase
(Pol I, II, or III in eukaryotic cells) to initiate transcription. The PIC often remains at
the promoter site, and is then available to initiate another round of transcription.
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•
TBP is a universal transcription factor, and is seen in all eukaryotes and archaea.
It sharply bends DNA at the TATA box.
Transcription elongation
•
Once initiation is complete and the open complex forms, the RNA polymerase
begins to read the template strand and add corresponding RNA nucleotides. This
process is not always efficient, and the polymerase may make several passes at
this. After a long enough RNA-DNA hybrid is made, the polymerase clears the
promoter region and moves rapidly downstream. This is preceeded by a large
conformational change in the polymerase core enzyme, as it clamps down on the
DNA and becomes quite processive.
•
While transcription elongation is quite rapid, the polymerase does not transcribe
all sequences with equal efficieny. The elongation rate is not uniform, and there
can be pausing or stalling. Elongation factors (GreA/GreB in bacterial systems;
TFIIS in eukaryotes) act to help the polymerase along by stimulating backtracking
and cleavage of the newly formed RNA (from the 3' end).
•
Other factors are involved in the elongation cycle - for example, in eukaryotic Pol
II there are the elongins and ELL proteins that increase the elongation rate, as
well as factors to remodel chromatin.
Fig. 12. Transcription elongation
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Transcription activation & repression
•
There are multiple modes of transcription activation. All these usually involve
different protein factors (activators) that bind DNA sequences (enhancer
sequences) in and around the promoter site. All these act to increase the affinity of
the initiation complex or RNA holoenzyme at the promoter (thus enhancing the
chance that a productive open complex will form and transcription will initiate).
•
When enhancer elements combine with poor promoter sequences, activators can
modulate the activity of a gene by several hundred or thousand fold. For example,
if a gene has a poor -35 or -10 box in its promoter site, it will be poorly expressed
- an activator protein can, in such a case, enhance the activity of the gene by
several orders of magnitude, by recruiting the RNA polymerase (or other
initiation factors) to the promoter site.
•
Repression, on the other hand, works by having protein (repressors) that sit on or
close to the promoter regions of the DNA, preventing RNA polymerase or
initiator/activator proteins from starting transcription initiation (thereby "turningoff" the gene).
•
Activators and repressors are usually DNA binding proteins. These proteins have
common DNA binding motifs such as Zn-fingers, helix-turn-helix, etc. Activators
also have regions that interact with different domains of the RNA polymerase
(parts of alpha, sigma, etc.).
•
In addition to protein activators, there are also DNA sequences that can directly
interact with RNA polymerase components. For example, the alpha subunit in the
bacterial polymerase has two domains - the alpha NTD (n-terminal domain) and
the alpha CTD (c-terminal domain), linked by a flexible linker region. The alpha
CTD can bind DNA sequences (UP elements) upstream of the promoter region,
enhancing the affinity of the polymerase on the promoter.
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Transcription termination
•
Termination in bacterial system can be broadly classified as "rho independent",
and "rho dependent".
o
In rho independent termination, there is formation of a stable GC rich
stem-loop in the newly synthesized RNA followed by a string of U's (A's
in the template strand) spaced about 20 bases downstream (these sites are
often called intrinsic terminators). The stem loop "snares" the polymerase,
slowing or stalling it. This pause, coupled with the low stability of the
RNA-DNA hybrid at the active site (run of A=U basepairs) allows the
RNA polymerase to fall off the template DNA and terminates the RNA
transcription for that gene.
o
In rho dependent termination, rho binds the newly formed RNA (as
hexamers), and stalls the RNA polymerase by interacting with it. In some
models, the Rho hexamer translocates on the RNA. Rho termination
activity is stimulated by ATP hydrolysis. This activity is greatly enhanced
by Nus factors.
•
In eukaryotic cells, transcription termination involves cleavage of the elongating
RNA chain by specific endonucleases which recognize particular sequences
(AAUAAA) in the newly formed RNA. Once this happens, the RNA elongation
complex is destabilized, and falls off the DNA. It is then available to attach to
another nearby PIC, and start transcribing again.
Fig. 13. Process of transcription in bacteria
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RNA processing (for eukaryotes)
Eukaryotic genes contain Exons (which encode information that will end up being
translated into protein) separated by Introns (sequences that will not encode proteins). At
the 5' end of a gene we find a promoter and often a CpG island. Both of these elements
regulate the transcription of a gene. At the 3' end of a gene we find a stop sequence, and
a signal for polyadenylation (AAUAAA). In Eukaryotes this primary RNA transcript is
then processed into a messenger RNA (mRNA) if the gene is to be translated into
protein. This involves removing introns, which do not encode for protein, and splicing
the remaining exons together. At the 5´ end of the mRNA a 7-Methyguanosine residue is
added to provide a protective cap. At the 3´ end of the mRNA, 50-100 adenosine
residues are added, generating a poly A tail.
REVERSE TRANSCRIPTION
Some viruses (such as HIV, the cause of AIDS), have the ability to transcribe RNA into
DNA. HIV has an RNA genome that is duplicated into DNA. The resulting DNA can be
merged with the DNA genome of the host cell. The main enzyme responsible for
synthesis of DNA from an RNA template is called reverse transcriptase. In the case of
HIV, reverse transcriptase is responsible for synthesizing a complementary DNA strand
(cDNA) to the viral RNA genome. An associated enzyme, ribonuclease H, digests the
RNA strand, and reverse transcriptase synthesizes a complementary strand of DNA to
form a double helix DNA structure. This cDNA is integrated into the host cell's genome
via another enzyme (integrase) causing the host cell to generate viral proteins that
reassemble into new viral particles. In HIV, subsequent to this, the host cell undergoes
programmed cell death, apoptosis of T cells. However, in other retroviruses, the host cell
remains intact as the virus buds out of the cell.
Some eukaryotic cells contain an enzyme with reverse transcription activity
called telomerase. Telomerase is a reverse transcriptase that lengthens the ends of linear
chromosomes. Telomerase carries an RNA template from which it synthesizes DNA
repeating sequence, or "junk" DNA. This repeated sequence of DNA is important
because, every time a linear chromosome is duplicated, it is shortened in length. With
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essential, repeated sequence rather than the protein-encoding DNA sequence farther away
from the chromosome end. Telomerase is often activated in cancer cells to enable cancer
cells to duplicate their genomes indefinitely without losing important protein-coding
DNA sequence. Activation of telomerase could be part of the process that allows cancer
cells to become immortal.
Translation: the Synthesis of Proteins
Role of mRNA
•
carries codons (3-nucleotide sequences) arranged in linear fashion that code for
amino acids
•
also carries signals needed to tell how to recognize ribosomes, start and stop
signals for decoding protein
•
leader sequence on small ribosome subunit binds to complementary sequence on
mRNA, allows initial formation of RNA-ribosome complex.
Fig. 14. Role of mRNA
Role of ribosome
•
two parts: a small subunit and a large subunit. These are separated except when
attached to m-RNA
•
ribosomes contain a set of ribosomal proteins and several types of ribosomal RNA
(r-RNA)
•
ribosomes catalyze the formation of peptide bonds; intially thought to be due to
protein activity (enzyme), but now known to be due to RNA catalytic activity
(ribozyme).
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Fig. 15. Ribosome
Role of tRNA
•
structure: 4 loops, anticodon, AA binding site
•
~ 60 types in bacteria (>100 in mammals)
•
only 73-93 nucleotides long
•
some bases modified after transcription; like pseudouridine.
•
extensive hairpin loops
•
anticodon site: recognizes codon on mRNA
•
Activation of tRNA: adding amino acids
o
requires special enzyme: AA-tRNA activating enzymes
o
ATP required, forms AA-AMP + PP, then AA-tRNA + AMP
Fig. 16. tRNA strcuture
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Initiation of Translation
•
small ribosomal subunit initiates binding to mRNA
•
locates 5' end of mRNA
•
small subunit ribosome finds first AUG codon = start codon
•
large ribosome binds
•
tRNA carries the amino acid methionine to first position
Fig. 17. Initiation of translation
Elongation of Translation
•
2 adjacent sites on ribosome: P (Peptide) and A (Amino Acid) site
•
A site accepts a new tRNA-AA
•
P site holds existing chain
•
peptide transferred from P site tRNA to A-site AA
•
enzyme activity is in ribosomal RNA (ribozyme)
•
also required: Energy (GTP) and elongation factors
Fig. 18. Elongation of translation
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Termination of Translation
•
reach a "stop codon" UAG, UAA, or UGA
•
no t-RNAs bind
•
instead, specific release factors required
•
Net cost of translation: 4 phosphate bonds/amino acid added.
Fig. 19. Termination of translation
NOTE: Typical RNA polymerization rate (~ 40 nts/second at 37 °C in bacteria - close to
translation rate of about ~15 aa/second).
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Fig. 20. The complete process of translation
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REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
Joint initiative of IITs and IISc – Funded by MHRD
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NPTEL – Biotechnology – Microbiology
Module 7 – Microbial Molecular Biology and Genetics
Lecture 5 - REGULATION OF GENES
GENE REGULATION
Each cell expresses, or turns on, only a fraction of its genes. The rest of the genes are
repressed, or turned off. The process of turning genes on and off is known as gene
regulation. Gene regulation also allows cells to react quickly to changes in their
environments. Gene regulation can occur at any point during gene expression, but most
commonly occurs at the level of transcription (when the information in a gene’s DNA is
transferred to mRNA). Signals from the environment or from other cells activate proteins
called transcription factors. These proteins bind to regulatory regions of a gene and
increase or decrease the level of transcription. By controlling the level of transcription,
this process can determine the amount of protein product that is made by a gene at any
given time.
Introduction
•
E. coli lives in colon. It has a metabolic pathway that allows for the synthesis of
the amino acid tryptophan (Trp).
o
This pathway starts with a precurser molecule and proceeds through five
enzyme catalyzed steps before reaching the final Trp product.
•
It is important that E. coli be able to control the rate of Trp synthesis because the
amount of Trp available from the environment varies considerably.
o
If we eat a meal with little or no Trp, the E. coli in our gut must
compensate by making more.
o
If we eat a meal rich in Trp, E. coli doesn't want to waste valuable
resources or energy to produce the amino acid because it is readily
available for use.
•
•
Therefore, E. coli uses the amount of Trp present to regulate the pathway.
o
If levels are not adequate, the rate of Trp synthesis is increased.
o
If levels are adequate, the rate of Trp synthesis is inhibited.
There are two ways of regulating the Trp pathway:
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o
The first method works to decrease the synthesis of Trp by inhibiting the
first enzyme in the pathway, preventing the rest of the pathway from
proceeding.
o
What inhibits the first enzyme? Trp does!

The more Trp in the cell, the more that can bind to the first enzyme
and prevent it from catalyzing the first step.
o
This method of regulation is feedback inhibition in which the end product
of a pathway acts as an inhibitor of an enzyme in that pathway.
•
The other method of control stops the production of the enzymes in the pathway
at the transcription level.
o
Remember that enzymes are proteins that must be transcribed and
translated from the genetic code.
o
If the genes for the enzymes are not transcribed to mRNA, then translation
to the enzymes cannot occur.
o
•
Without enzymes, there is no Trp synthesis.
This method of control is called regulation of gene expression because control is
taking place at the genetic level. This method of control will now be examined in
detail.
Trp Operon:
Fig. 21. Structure of trp operon
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•
The genes for the five enzymes in the Trp synthesis pathway are clustered on the
same chromosome in what is called the Trp operon.
•
The Trp operon has three components:
o
Five Structural Genes:

These genes contain the genetic code for the five enzymes in the Trp
synthesis pathway
o
One Promoter:

o
DNA segment where RNA polymerase binds and starts transcription
One Operator:

DNA segment found between the promoter and structural genes. It
determines if transcription will take place. If the operator is turned
"on", transcription will occur.
•
When nothing is bonded to the operator, the operon is "on".
o
RNA polymerase binds to the promoter and transcription is initiated.
o
The five structural genes are transcribed to one mRNA strand.
o
The mRNA will then be translated into the enzymes that control the Trp
synthesis pathway.
•
The operon is turned "off" by a specific protein called the repressor.
o
The repressor is a product of the regulator gene which is found some
distance from the operon.

Transcription of the regulator produces mRNA which is translated
into the repressor.

The repressor is inactive in this form and cannot bind properly to the
operator with this conformation.
o
To become active and bind properly to the operator, a co-repressor must
associate with the repressor.

The co--repressor for this system is Trp

This makes sense because E. coli does not want to synthesize Trp if it
is available from the environment

The more Trp available, the more that can associate with repressor
molecules.
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o
An active repressor binds to the operator blocking the attachment of RNA
polymerase to the promoter.
o
Without RNA polymerase, transcription and translation of the structural
genes can't occur and the enzymes needed for Trp synthesis are not made.
Repressible vs Inducible Systems
•
The Trp pathway is anabolic as Trp is being synthesized. The Trp and other
regulated anabolic pathways are usually repressible because the system can be
repressed by an overabundance of the end product.
o
The end product, Trp, in this case, decreases or stops the transcription of
the enzymes necessary for its production.
•
Regulated catabolic pathways, on the other hand, are usually inducible because
the pathway is stimulated rather than inhibited by a specific molecule. An
example of an inducible system is lactose metabolism.
Fig. 22. Inducible and repressible operons
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The lac Operon
•
The genes that code for the enzymes needed for lactose catabolism are clustered
on the same chromosome in what is called the lac operon.
•
The Lac operon has three components:
o
Three Structural Genes:

These contain the genetic code for the three enzymes in the lac
catabolic pathway
o
One Promoter:

o
DNA segment where RNA polymerase binds and starts transcription
One Operator:

DNA segment found between the promoter and structural genes.

It determines if transcription will take place.

If the operator in turned "on", transcription will occur.
Fig. 23. The lac operon
•
As in the Trp operon, the Lac operon is turned "off" by a specific protein called
the repressor.
o
The repressor is the product of the regulator gene which is found outside
the operon.

Transcription of the regulator produces mRNA which is translated
into the repressor.
o
But unlike the Trp operon, the repressor is active in this form and does not
require a co-repressor.
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o
The active repressor binds to the operator blocking the advancement of
RNA polymerase to the structural genes.
o
Without RNA polymerase, transcription and translation of the genes can't
occur and the enzymes needed for Lac metabolism are not made.
•
What turns the Lac operon "on"? Lactose does!
o
This makes sense because the cell only needs to make enzymes to
catabolize lactose if lactose is present.
•
When lactose enters the cell, allolactose, an isomer of lactose is formed.
•
Allolactose binds to the repressor and alters its conformation so that it can't bind
to the operator.
•
RNA polymerase can now start transcription.
o
The three structural genes are transcribed to one mRNA strand.
o
The mRNA will then be translated into the enzymes that control lactose
catabolism.
•
In this sense, allolactose is an inducer.
Fig. 24. The lac operon: a model of gene regulation in prokaryotic cells
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Negative vs Positive Control
•
While the Trp operon is an example of repressible gene regulation and the
Lac operon is an example of inducible gene regulation, both are examples
of negative control of genes because both operons are shut "off" by an
active repressor.
•
Gene regulation would be positive; on the other hand, if an activator
molecule turned the operon "on".
•
The Lac operon is also an example of a positive control system and is
turned on by the cAMP-CAP complex, as described below:
•
E. coli can be described as a fussy eater.
o
Its first choice at every meal is glucose because glucose supplies
maximum energy for growth.
o
Therefore, E. coli will only metabolize lactose if concentrations of
glucose are low.
•
For this to work, there must be a signal to tell the Lac operon that glucose
is not available and to start transcribing the genes to metabolize lactose.
o
This signal is a small molecule called cyclic AMP (cAMP).

The amount of cAMP present in a cell is inversely
proportional to the amount of glucose present.

As a result, the absence of glucose results in an increase in
cAMP in the cell.
•
The following describes the situation where there is lactose but no glucose
available to the cell:
o
No glucose means high levels of cAMP.
o
cAMP binds to a molecule known as CAP.
o
CAP, when in association with cAMP, can bind to the promoter at
the CAP binding site.
o
Here, the cAMP-CAP complex stimulates transcription by helping
RNA polymerase bind to the promoter.

RNA polymerase has a weak affinity for the Lac promoter
and will not bind without this help.
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o
Remember with lactose present so is allolactose.

Allolactose binds to the repressor and prevents it from
binding to the operator.

Therefore, transcription and translation of the genes can
occur.
o
The following depicts what happens when glucose and lactose are
both present for E. coli to metabolize:

With glucose present, there is very little or no cAMP.

It cannot bind to the CAP binding site.

Without this complex, RNA polymerase cannot bind to the
promoter and transcription cannot occur.

Even though allolactose is present and blocks the action of
the repressor, there is no transcription of the lac genes
because glucose is present.
Fig. 25. Overall structural elements of Lac Operon
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Fig. 26. Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized
Fig. 27. Lactose present, glucose present (cAMO level low): little lac mRNA synthesized
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REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
Other References:
1. http://www.nature.com/scitable/topic/gene-expression-and-regulation-15
2. http://ghr.nlm.nih.gov/handbook/howgeneswork/geneonoff
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Module 7 – Microbial Molecular Biology and Genetics
Lecture 6 – DNA Repair and Microbial Recombination
DNA REPAIR
It can be defined as “any one of the cellular process that attempts to correct errors in
cellular DNA which occurred during cell division or by external environment”.
There are many cellular proteins and enzymes which help in repairing most of the errors
by means of different mechanisms.
Photoreactivation
•
Catalyzed by DNA-Photolyase (DPL)
•
Reverses cyclobutyl pyrimidine dimers resulting from UV irradiation.
•
It has low abundance (10 to 20 molecules per cell) in E. coli.
•
DPL binds irradiated DNA 100x better than non-irradiated DNA.
•
DPL contains a non-covalently associated chromophore (FADH or FADH2).
•
The photochemical mechanism of photorepair has been proposed to be lightdependent redox reaction between the singlet excited state of FADH2 and the
pyrimidine dimer.
•
Photolyase has not yet been identified in placental mammals.
Fig. 28. Photoreactivation
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Aberrant Methylation
•
Catalyzed by 6-O-methylguanine methyltransferase (6-O-MGM)
•
6-O-MGM recognizes 6-O-methylguanine in DNA and removes the methyl
group, transferring the group to an amino acid on itself in a "suicide" mechanism.
•
6-O-MGM is encoded by the ada gene in E. coli and the MGMT gene in
eukaryotes.
•
Mice knocked out for MGMT are cancer-prone and sensitive to methylation
agents
Common Features of Excision-Type Repair Pathways
•
Recognition: Altered DNA is recognized and bound by a specific damagerecognition protein. This first step recruits other components required for the
repair reaction.
•
Excision: Damaged base(s), and in some cases adjacent nucleotides, are excised
from the strand by exonucleases, resulting in a gapped DNA.
•
Resynthesis: The gap is refilled by a DNA polymerase using the complementary
strand as a template.
Base-Excision Repair (BER)
•
Damaged bases are removed as free bases.
•
BER primarily handles oxidative and alkylative damage.
•
BER is thought to have an important role in aging.
•
BER recognizes base deamination, oxidative damage, and other minor base
modifications.
•
Five gene products are required for BER: glycosylase, AP endonuclease,
phosphodiesterase, DNA polymerase, and DNA ligase.
•
DNA glycosylase recognizes the damaged base and removes it, generating an AP
(apurinic, apyramidinic) site.
•
AP endonuclease cleaves the phosphodiester bond, generating a single-strand
break with a 5'-terminal deoxyribophosphate moiety.
•
The 5'-deoxyribophosphate is excised by action of a DNA phosphodiesterase.
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•
The resulting single-nucleotide gap is repaired by DNA polymerase β (beta).
•
The resulting nick is sealed by DNA ligase.
•
BER is relatively inefficient, due to the large number of peptides needed to
recognize each damage type.
Nucleotide-Excision Repair (NER)
•
Damaged bases are removed as oligonucleotides.
•
NER is primarily responsible for removal of UV-induced damage and bulky
adducts, but also removes ~20% of oxidative damage.
•
Deficiencies cause many human disorders.
•
Xeroderma Pigmentosum (both classical and variant) and Cockayne's syndrome
are caused by defects in NER, resulting in various detrimental effects upon
exposure to UV light.
Fig. 29. a) Nucleotide excision repair; b) base excision repair
DNA Mismatch Repair (MMR)
•
Mismatches are removed as long oligonucleotides.
•
MMR is primarily responsible for removal of replicative errors.
•
Also prevents recombination of non-homologous sequences
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•
Deficient in many human cancers.
•
MMR recognizes base-base mismatch and small insertion/deletion loops.
Fig. 30. DNA mismatch repair
Error prone translation DNAsynthesis
When this pathway is active, DNA repair becomes significantly less accurate and a high
mutation rate occurs. It is a part of cellular stress response to extensive DNA damage,
also known as SOS response. The cell, at all cases had to survive, so DNA gets repaired,
although it contains a lot of errors.
DNA RECOMBINATION
DNA recombination involves the exchange of genetic material either between multiple
chromosomes or between different regions of the same chromosome. This process is
generally mediated by homology; that is, homologous regions of chromosomes line up in
preparation for exchange, and some degree of sequence identity is required.
•
Homologous recombination occurs between two homologous chromosomes.
•
Nonhomologous, or illegitimate recombination occurs between two different
chromosomes, though the segment at recombination sites may be related.
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•
Site-specific recombination can result in integration of viral, bacterial, or plasmid
DNA into a chromosome at a specific location (such as att).
•
Replicative recombination results in sequence transposition, and is mediated by
transposase enzymes.
Types of Homologous Recombination
•
Reciprocal recombination results from two chromosomes exchanging the same
amount of DNA.
•
Non-reciprocal recombination results from two chromosomes exchanging
different amounts of DNA.
•
Intramolecular recombination results from recombination in chromosomal loops.
o Direct repeats cause plasmid formation from the intervening loop.
o Inverted repeats cause the intervening loop to change direction.
•
Double crossover results in exchanging a short segment of DNA, rather than a
large segment of chromosome from the crossover point to the terminus.
Holliday Model for Homologous Recombination
•
Two DNA molecules with nicks induced by an endonuclease in strands of the
same polarity can invade each other with free single strands.
•
A Holliday junction, or Chi structure, is formed as a recombination intermediate,
and is solidified by ligase sealing the nicks, uniting the two homologues.
•
Branch migration allows exchange of material, since the loose area around the
junction allows unzipping/rezipping.
•
New nicks allow separation of recombined DNA and resolution of the
recombination intermediate.
•
The Holliday model assumes reciprocal and equal exchange of genetic material
between DNA molecules.
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Fig. 31. Holliday model
Double-Strand Break Repair Model (Szostak Model) for Recombination
•
Double-strand breaks are introduced into one of the homologues.
•
Broken ends are recessed by specific exonucleases which produce longer 3' single
strands.
•
The single strands can invade a region of homologous DNA.
•
The invading end serves as a primer for a polymerase which extends it,
unwidnding the template DNA and generating a D-loop. A Holliday junction is
formed, and DNA synthesis proceeds until it reaches the opposing end of the
recombining chromosome.
•
The displaced DNA of the opposing end of the invading chromosome anneals to
the template (invaded chromosome), forming a second Holliday junction by
ligation of the strands. Holliday junctions can migrate prior to resolution with
resolvase endonucleases.
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•
Resolution can cut the invading strands, resulting in a non-crossover event, since
each chromosome has only a tiny fragment of DNA from the other chromosome.
•
Crossover results from cutting the strands not participating in the recombination,
generating a crossover by leaving a large segment of each chromosome attached
to the other.
•
The Szostak model accounts for non-reciprocal recombination.
Fig. 32. Double and single strand break repair model
Factors Involved in Each Recombination Step
•
The Szostak model is an error-free pathway (since it can only occur in
homologues), and is of major importance in yeasts and mammals.
•
A 5' to 3' recession reaction is mediated by endonucleases.
•
Strand invasion requires multiple Rad proteins, including DNA helicases and
DNA end-binding proteins.
•
New DNA synthesis requires DNA Pol δ and ε (delta and epsilon).
Repair of DSBs by Non-Homologous End Joining (NHEJ)
•
NHEJ is an error-prone pathway, and is the major pathway in mammals for repair
of DSBs.
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•
DNA-PKcs (DNA protein kinase, catalytic subunit) binds ends of the DSB
(double-strand break).
•
Endonucleases create blunt (no overhang) ends.
•
Synapsis is achieved through microhomologies.
•
Ends are ligated, and, due to end-blunting and other factors, small insertions and
deletions are introduced. The errors are often of little consequence, since most
DNA is non-coding.
Site specific recombination
Site-specific recombination, also known as conservative site-specific recombination,
is a type of genetic recombination in which DNA strand exchange takes place between
segments
possessing
only
a
limited
degree
of sequence
homology. Site-
specific recombinases perform rearrangements of DNA segments by recognizing and
binding to short DNA sequences (sites), at which they cleave the DNA backbone,
exchange the two DNA helices involved and rejoin the DNA strands. While in some sitespecific recombination systems just a recombinase enzyme and the recombination sites is
enough to perform all these reactions, in other systems a number of accessory proteins
and/or accessory sites are also needed.
Site-specific recombination systems are highly specific, fast and efficient, even when
faced with complex eukaryotic genomes. They are employed in a variety of cellular
processes, including bacterial genome replication, differentiation and pathogenesis, and
movement of mobile genetic elements. For the same reasons, they present a potential
basis for the development of genetic engineering tools.
Recombination sites are typically between 30 and 200 nucleotides in length and consist
of two motifs with a partial inverted-repeat symmetry, to which the recombinase binds,
and which flank a central crossover sequence at which the recombination takes place. The
pairs of sites between which the recombination occurs are usually identical, but there are
exceptions (e.g. attP and attB of λ integrase,)
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Fig. 33. Insertion and excision mediated by aligned Lox sites and the cre recombinase. Red X designates recombination.
REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
Other References:
1. http://www.nature.com/scitable/topicpage/genetic-recombination-514
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Module 7 – Microbial Molecular Biology and Genetics
Lecture 7 –Bacterial Plasmids and Transposable elements
PLASMIDS
It is an extra chromosomal DNA molecule separate from the chromosomal DNA
which is able to replicate independently of the chromosomal DNA. Most commonly
found as small circular, double-stranded DNA molecules in bacteria, plasmids are
sometimes present in archaea and eukaryotic organisms. In nature, plasmids carry genes
that may benefit survival of the organism (e.g. antibiotic resistance), and can frequently
be transmitted from one bacterium to another (even of another species) via horizontal
gene transfer. Artificial plasmids are widely used as vectors in molecular cloning, serving
to drive the replication of recombinant DNA sequences within host organisms.
•
They vary from 1kb to 1000kb
•
The term plasmid was first introduced by the American molecular biologist
Joshua Lederberg in 1952.
•
The plasmid does not contribute to the genome of the bacterial cell it is present in
but often translates proteins of significance. For example
o
It contains the antibiotic resistance gene that helps the bacteria survive
from that specific antibiotic.
o
It can provide the bacteria with an ability to fix elemental nitrogen or to
degrade calcitrant organic compounds which provide an advantage under
conditions of nutrient deprivation.
Fig. 34. This image shows a line drawing of a bacterium with its chromosomal DNA and several plasmids within it.
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Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools
in genetics and biotechnology labs, where they are commonly used to multiply (make
many copies of) or express particular genes. Many plasmids are commercially available
for such uses. The gene to be replicated is inserted into copies of a plasmid containing
genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS,
or polylinker), which is a short region containing several commonly used restriction sites
allowing the easy insertion of DNA fragments at this location. Next, the plasmids are
inserted into bacteria by a process called transformation. Then, the bacteria are exposed
to the particular antibiotics. Only bacteria that take up copies of the plasmid survive,
since the plasmid makes them resistant. In particular, the protecting genes are expressed
(used to make a protein) and the expressed protein breaks down the antibiotics. In this
way, the antibiotics act as a filter to select only the modified bacteria. Now these bacteria
can be grown in large amounts, harvested, and lysed (often using the alkaline lysis
method) to isolate the plasmid of interest. Another major use of plasmids is to make large
amounts of proteins. In this case, researchers grow bacteria containing a plasmid
harboring the gene of interest. Just as the bacterium produces proteins to confer its
antibiotic resistance, it can also be induced to produce large amounts of proteins from the
inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it
then codes for, for example, insulin or even antibiotics. A plasmid can contain inserts of
up to 30-40 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes
deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are
used.
Replication of plasmid
Non Integrative Replication
In this type of replication the plasmid DNA once introduced into the cell grows as per the
copy number and the multiplication of the cells
Integrative Plasmid
Episome
Under certain conditions some plasmids may integrate into the bacterial chromosome.
They are called episome or integrative plasmids. At this stage they replicate along with
the bacterial chromosome.
The plasmids in this way are classified into 2 types
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Relaxed plasmids
They are the ones which are normally maintained at multiple copies per cell.
Stringent plasmids
They are the ones which have a limited number of copies per cell.
In this case of plasmid replication, the plasmid DNA is integrated in the bacterial
chromosome and grows along with the cell. It uses the bacterial machinery for division.
A good example of this type of replication is Ti Plasmid, often used in agricultural
genetic engineering experiments. It completely uses the cell genetic mechanism to grow.
Fig. 35. Replication of plasmid
Types of plasmid
•
Fertility plasmid:
That contains the tra genes required for conjugation else known as F plasmids.
For example F plasmids of E coli.
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Fig. 36. Fertility plasmid
•
Resistance Plasmid or R plasmids
Carry genes of resistance to one or more antibacterial agents such as ampicilin,
etc
•
Col plasmid:
It contains genes for production of bacteriocins, proteins that kill other bacteria
example col E1.
•
Virulent plasmids:
Which in turn convert bacteria into a pathogen. For example, the Ti plasmid of
Agrobacterium tumefaciens induces crown gal disease on dicot of the palnts
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Table 2. Size of some plasmids as vectors
Plasmid
Size
Mol. Wt
Marker
Organism
Apr
Escherichia coli K12
in kb
puc 8
2.1
2.7
JM83
col E1
6.4
6.6
rP4
54.0
56.4
E1 imm
Escherichia coli JC411
Apr, Kmr, Escherichia coli K12 J53
Tcr
ToL
117.0
78.0
pseudomonas
pTiach 5
213.0
142.0
A. tumefaciens
TRANSPOSABLE ELEMENTS
A transposable element (TE) is a DNA sequence that can change its relative position
(self-transpose) within the genome of a single cell. Barbara McClintock's discovery of
these jumping genes early in her career earned her a Nobel prize in 1983. They are often
considered "junk DNA”.
Fig. 37. Bacterial composite transposon
Transposable elements are only one of several types of mobile genetic elements. They are
assigned to one of two classes according to their mechanism of transposition, which can
be described as either “copy or paste" (for class I TEs) or "cut and paste" (for class II
TEs).
Class I (retrotransposons): They copy themselves in two stages, first from DNA
to RNA by transcription, then from RNA back to DNA by reverse transcription. The
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DNA copy is then inserted into the genome in a new position. Reverse transcription is
catalyzed by a reverse transcriptase, which is often coded by the TE itself.
Retrotransposons behave very similarly to retroviruses, such asHIV.
There are three main orders of retrotransposons (other orders are less abundant):

Those with long terminal repeats (LTRs): encode reverse transcriptase, similar to
retroviruses;

LINEs: encode reverse transcriptase, lack LTRs, transcribed by RNA polymerase II;

SINEs: do not code for reverse transcriptase, transcribed by RNA polymerase III.
Retroviruses can be considered as TEs. Indeed, after entering a host cell and converting
their RNA into DNA, retroviruses integrate this DNA into the DNA of the host cell. The
integrated DNA form (provirus) of the retrovirus is viewed as a particularly specialized
form of eukaryotic retrotransposon, which is able to encode RNA intermediates that
usually can leave the host cells and infect other cells. The transposition cycle of
retroviruses also has similarities to that of prokaryotic TEs. The similarities suggest a
distant familial relationship between these two TEs types.
Class II (DNA transposons): By contrast, the cut-and-paste transposition mechanism of
class II TEs does not involve an RNA intermediate. The transpositions are catalyzed by
various types oftransposase enzymes. Some transposases can bind non-specifically to any
target site, while others bind to specific sequence targets. The transposase makes a
staggered cut at the target site producing sticky ends, cuts out the DNA transposon and
ligates it into the target site. A DNA polymerase fills in the resulting gaps from the sticky
ends and DNA ligase closes the sugar-phosphate backbone. This results in target site
duplication and the insertion sites of DNA transposons may be identified by short direct
repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by
inverted repeats (which are important for the TE excision by transposase). The
duplications at the target site can result in gene duplication, which plays an important role
in evolution.
Cut-and-paste TEs may be duplicated if transposition takes place during S phase of
the cell cycle when the "donor" site has already been replicated, but the "target" site has
not.
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Both classes of TEs may lose their ability to synthesise reverse transcriptase or
transposase through mutation, yet continue to jump through the genome because other
TEs are still producing the necessary enzymes. Hence, they can be classified as either
"autonomous" or "non-autonomous". For instance for the class II TEs, the autonomous
ones have an intact gene that encodes an active transposase enzyme; the TE does not
need another source of transposase for its transposition. In contrast, non-autonomous
elements encode defective polypeptides and accordingly require transposase from another
source.
REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
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Module 7 – Microbial Molecular Biology and Genetics
Lecture 8 – Mutations and their chemical basis, detection and isolation
of mutants
MUTATIONS
•
A Mutation occurs when a DNA gene is damaged or changed in such a way as to
alter the genetic message carried by that gene.
•
A Mutagen is an agent of substance that can bring about a permanent alteration to
the physical composition of a DNA gene such that the genetic message is
changed.
•
Once the gene has been damaged or changed the mRNA transcribed from that
gene will now carry an altered message.
•
The polypeptide made by translating the altered mRNA will now contain a
different sequence of amino acids. The function of the protein made by folding
this polypeptide will probably be changed or lost.
Chemical Mutagens – They change the sequence of bases in a gene in a number of
ways:
•
Mimic the correct nucleotide bases in a DNA molecule, but fail to base pair
correctly during DNA replication.
•
Remove parts of the nucleotide (such as the amino group on adenine), again
causing improper base pairing during DNA replication.
•
Add hydrocarbon groups to various nucleotides, also causing incorrect base
pairing during DNA replication.
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Radiation - High energy radiation from a radioactive material or from X-rays is
absorbed by the atoms in water molecules surrounding the DNA. This energy is
transferred to the electrons which then fly away from the atom. Left behind is a free
radical, which is a highly dangerous and highly reactive molecule that attacks the DNA
molecule
and
alters
it
in
many
ways.
Radiation can also cause double strand breaks in the DNA molecule, which the cell's
repair mechanisms cannot put right.
Sunlight- It contains ultraviolet radiation (the component that causes a suntan) which,
when absorbed by the DNA causes a cross link to form between certain adjacent bases. In
most normal cases the cells can repair this damage, but unrepaired dimers of this sort
cause the replicating system to skip over the mistake leaving a gap, which is supposed to
be
filled
in
later.
Unprotected exposure to UV radiation by the human skin can cause serious damage and
may lead to skin cancer and extensive skin tumors.
Spontaneous - Mutations occur without exposure to any obvious mutagenic agent.
Sometimes DNA nucleotides shift without warning to a different chemical form (know as
an isomer) which in turn will form a different series of hydrogen bonds with it's partner.
This leads to mistakes at the time of DNA replication.
•
EXAMPLES :
1. Nitrous Acid:

Nitrous Acid affects DNA complementation.

The acid randomly modifies the base adenine so that it will pair
with cytosine instead of thymine.

This change is made evident during DNA replication when a new
base pair appears in daughter cells in a later generation.
2. A Base Analog:

A base analog is a compound sufficiently similar to one of the four
DNA bases but have different pairing properties.
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
For example, 5-bromouracil is the analog of thymine but
sometimes pairs with guanine and 2-aminopurine is the analog of
adanine but sometimes pairs with cytosine.

The incorporation of a base analog will result in base pair
substitution .
3. UV Light:

Exposure to direct UV light induces covalent linking between
adjacent thymine nucleotides on a DNA strand forming a thymine
dimer.

These dimers cause the strand to buckle, disrupting normal base
pairing. This prevents proper replication and transcription.

Bacteria have enzymes to fix the damage created by UV light.

An enzyme cuts the DNA at two point and removes the damaged
portion.

DNA polymerase synthesizes a new DNA segment using the
healthly strand as a template.

DNA ligase joins the new fragment to the old strand.
Types of Mutations:
NOTE: For all others examples cited below, the below given DNA sequence shall be
considered as wild type:

ATG CCG TGT CAG ATG TTC

AUG CCG UGU CAG AUG UUC
Met
Pro Cys Gln
met
---------------- DNA
------------ mRNA
phe ------ Amino acid sequence
1. Synonymous / Silent Mutations
1. No alteration in polypeptide product of the gene
2. Single base pair substitution
3. Occur in the third position of a codon
4. Codes for the same amino acid
5. No alteration of the protein
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Example: Samesense Mutation:
a codon is changed to a different codon that specifies the same amino acid.
ATG CCG TGC CAG ATG TTC
-----MUTATED DNA (compare with wild type)
AUG CCG UGC CAG AUG UUC ----- mRNA
Met
Pro Cys
Gln
Met Phe -------- amino acid sequence
2. Non-Synonymous Mutations
1. Occur less frequently than synonymous mutations
2. Leads to alteration in the encoded polypeptide
3. Result in abnormal function→ disease
Occur in one of three main ways:
1. Missense
2. Nonsense
3. Frameshift
Missence mutation: a codon is changed to a different codon that specifies a different
amino acid.
ATG CCG TGG CAG ATG TTC
-----MUTATED DNA
AUG CCG UGG CAG AUG UUC ----- mRNA
Met
Pro Trp
Gln
Met Phe -------- amino acid sequence
Nonsense Mutation: A codon that specifies an amino acid is changed to a stop codon.
(termination codon), this mutation usually destroys the function of the gene product.
ATG CCG TGA CAG ATG TTC
----- MUTATED DNA
AUG CCG UGA CAG AUG UUC
-----
Met Pro STOP
mRNA
-------- amino acid sequence
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Frameshift Mutations: One or 2 nucleotide pairs are inserted into or deleted from the
molecule, causing an alteration of the reading frame. As the result of this shift, codons
downstream of the insertion or deletion site specify an entirely new sequence of amino
acids. Depending on where the insertion or deletion occurs in the gene, different effects
can be generated. In addition to producing an entirely new polypeptide sequence
immediately after the change, frameshift mutations usually produce a stop or termination
codon within a short distance of the mutation. This codon terminates the already altered
polypepetide chain. A frame shift in a gene specifying an enzyme usually result in a loss
of enzyme activity. If the enzyme is an essential one, the effect on the organism can be
disastrous.
Fig. 38. Types of mutations
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Mutation in non-coding DNA
They will have a phenotyping effect if it occurs in
1. Regulatory elements (e.g. TATA box) → affect level of gene expression
2. Splicing of introns: highly conserved GT & AG at the end of introns
Either:
a.
coding sequences being lost
b. intronic sequences being added to the mRNA
Functional effects of Mutation on the Protein
1. Loss of function
2. Gain of Function
Loss of Function
 Reduce in activity
In heterzygous state →half normal levels of the protein product
 Or complete loss of the gene product
Gain of Function:
 Increased levels of gene expression
 Development of a new function(s) of the gene product
Chromosomal Mutations:
Chromosomal mutations are grouped into four broad types:
1)
Deletion: are chromosomal changes in which one or more genes or segments of
chromosomal DNA are lost.
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2)
Duplication: are chromosomal changes in which one copy or more copies of a
gene are present on the same chromosome
3) Inversion: in which a segment of DNA is released and rotated 180 degrees before
being reinserted into the DNA. If the inverted DNA segment carries part of the proteincoding sequence, the resulting protein would be drastically altered and most likely
nonfunctional.
4) Translocation: occur when a segment of DNA moves from one chromosome and
inserted into a different nonhomologous chromosome. Translocation can also be
reciprocal, that is two nonhomologous chromosome may break and trade pieces of DNA.
Translocation mutations frequently cause problems in meiosis and sometimes lead to
aneuploidy (the gain or loss of chromosome)
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Mutant Isolation
•
How can you tell if there are any mutant colonies in a culture? This is done by
either positive (direct) selection or by negative (indirect) selection.
1. Positive Selection:

Positive selection entails growing the culture on a medium that will
allow for the growth of only the mutant colonies.

If, for example, we want to find mutants that resistant to penicillin,
we grow the culture on a medium that contains penicillin. Only
those colonies that are resistant to penicillin will grow and we can
identify them directly.
Fig. 39. Mutant isolation
2. Negative Selection:

Negative selection is used to identify mutants that have lost the
ability to perform a certain function that their parents had.

Auxotrophic mutants, for example, are bacteria that have
lost the ability to synthesize an essential nutrient.
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
The replica-plating technique is used to identify mutants by
negative selection.

the replica-plating technique can be used, for example, to
identify mutants that have lost the ability to synthesize the
amino acid histidine. Therefore, mutants are His- and
require histidine in order to survive.

Inoculate a histidine enriched medium with
bacteria. Incubate so that cells can form colonies.
This is the master plate.

Press a sterile velvet surface into the colonies of the
master plate. Some cells from each of the colonies
adhere to the velvet.

Prepare two mediums, one with histidine, the other
without histidine.

Transfer cells from the velvet to each plate.

Compare growth on the two plates after incubation.
Colonies that grow on the histidine enriched
medium but not on the medium lacking histidine are
His- mutants
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Fig. 40. Negative selection
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Fig. 41. Ames test
The Ames Test utilizes a histidine auxotroph of Salmonella to determine if a
chemical agent is a mutagen. Though some spontaneous back mutations (a reversion
back to the strain of Salmonella that can synthesize histadine) can occur, if many colonies
of the microbe appear on the minimal plate after the addition of the test chemical, this is
an indication the the chemical is a mutagen.
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REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
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Module 7 – Microbial Molecular Biology and Genetics
Lecture 9 - BACTERIAL CONJUGATION, TRANSDUCTION AND
TRANSFORMATION
Sometimes when two pieces of DNA come into contact with each other, sections
of each DNA strand will be exchanged. This is usually done through a process called
crossing over in which the DNA breaks and is attached on the other DNA strand leading
to the transfer of genes and possibly the formation of new genes. Genetic recombination
is the transfer of DNA from one organism to another. The transferred donor DNA may
then be integrated into the recipient's nucleoid by various mechanisms. In the case of
homologous recombination, homologous DNA sequences having nearly the same
nucleotide sequences are exchanged by means of breakage and reunion of paired DNA
segments. Genetic information can be transferred from organism to organism through
vertical transfer (from a parent to offspring) or through horizontal transfer methods such
as conjugation, transformation or transduction. Bacterial genes are usually transferred to
members of the same species but occasionally transfer to other species can also occur
Horizontal gene transfer, also known as lateral gene transfer, is a process in
which an organism transfers genetic material to another organism that is not its
offspring. The ability of Bacteria and Archaea to adapt to new environments as a part of
bacterial evolution most frequently results from the acquisition of new genes through
horizontal gene transfer rather than by the alteration of gene functions through
mutations. (It is estimated that as much as 20% of the genome of Escherichia
coli originated from horizontal gene transfer.)
Horizontal gene transfer is able to cause rather large-scale changes in a bacterial
genome. For example, certain bacteria contain multiple virulence genes called
pathogenicity islands that are located on large, unstable regions of the bacterial genome.
These pathogenicity islands can be transmitted to other bacteria by horizontal gene
transfer. However, if these transferred genes provide no selective advantage to the
bacteria that acquire them, they are usually lost by deletion. In this way the size of the
bacterium's genome can remain approximately the same size over time.
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There are three mechanisms of horizontal gene transfer in bacteria: transformation,
transduction, and conjugation. The most common mechanism for horizontal gene
transmission among bacteria, especially from a donor bacterial species to different
recipient species, is conjugation. Although bacteria can acquire new genes through
transformation and transduction, this is usually a more rare transfer among bacteria of the
same species or closely related species.
CONJUGATION
Bacterial conjugation is the transfer of genetic material between bacterial cells
by direct cell-to-cell contact or by a bridge-like connection between two cells.
Discovered in 1946 by Joshua Lederberg and Edward Tatum, conjugation is a mechanism
of horizontal gene transfer as are transformation and transduction although these two
other mechanisms do not involve cell-to-cell contact.
Fig. 42. Genetic recombination
Lederberg and Tatum did not directly prove that physical contact of the cells was
necessary for gene transfer. This evidence was provided by Bernard Davis (1950), who
constructed the U tube consisting of two pieces of curved glass tubing fused at the base to
form a U shape with a fritted glass filter between the halves. The filter allows the passage
of media and not bacteria. Davis discovered that when two auxotrophic strains were
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separated by the filter, gene transfer could not take place. Therefore direct contact is
necessary for the recombination to take place as discovered by Lederberg and Tatum.
Fig. 43. U-tube experiement
Bacterial conjugation is often regarded as the bacterial equivalent of sexual
reproduction or mating since it involves the exchange of genetic material. During
conjugation the donor cell provides a conjugative or mobilizable genetic element that is
most often a plasmid or transposon. Most conjugative plasmids have systems ensuring
that the recipient cell does not already contain a similar element.
The genetic information transferred is often beneficial to the recipient. Benefits
may
include antibiotic
resistance, xenobiotic tolerance
or
the
ability
to
use
new metabolites. Such beneficial plasmids may be considered bacterial endosymbionts.
Other elements, however, may be viewed as bacterial parasites and conjugation as a
mechanism evolved by them to allow for their spread.
The prototypical conjugative plasmid is the F-plasmid, or F-factor. The Fplasmid
is
an episome (a
plasmid
that
can
integrate
itself
into
the
bacterial chromosome by homologous recombination) with a length of about 100 kb. It
carries its own origin of replication, the oriV, and an origin of transfer, or oriT. There can
only be one copy of the F-plasmid in a given bacterium, either free or integrated, and
bacteria that possess a copy are called F-positive or F-plus (denoted F+). Cells that lack F
plasmids are called F-negative or F-minus (F-) and as such can function as recipient cells.
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Fig. 44. Conjugation
Among other genetic information the F-plasmid carries a tra and trb locus, which
together are about 33 kb long and consist of about 40 genes. The tra locus includes
the pilin gene and regulatory genes, which together form pili on the cell surface. The
locus also includes the genes for the proteins that attach themselves to the surface of Fbacteria and initiate conjugation. Though there is some debate on the exact mechanism
of conjugation it seems that the pili are not the structures through which DNA exchange
occurs. This has been shown in experiments where the pilus are allowed to make contact,
but then are denatured with SDS and yet DNA transformation still proceeds. Several
proteins coded for in the tra or trb locus seem to open a channel between the bacteria and
it is thought that the traD enzyme, located at the base of the pilus, initiates membrane
fusion.
When conjugation is initiated by a signal the relaxase enzyme creates a nick in
one of the strands of the conjugative plasmid at the oriT. Relaxase may work alone or in a
complex of over a dozen proteins known collectively as a relaxosome. In the F-plasmid
system the relaxase enzyme is called TraI and the relaxosome consists of TraI, TraY,
TraM and the integrated host factor IHF. The nicked strand, or T-strand, is then unwound
from the unbroken strand and transferred to the recipient cell in a 5'-terminus to 3'terminus direction. The remaining strand is replicated either independent of conjugative
action (vegetative replication beginning at the oriV) or in concert with conjugation
(conjugative replication similar to the rolling circle replication of lambda phage).
Conjugative replication may require a second nick before successful transfer can occur. A
recent report claims to have inhibited conjugation with chemicals that mimic an
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intermediate step of this second nicking event. If the F-plasmid that is transferred has
previously been integrated into the donor’s genome some of the donor’s chromosomal
DNA may also be transferred with the plasmid DNA. The amount of chromosomal DNA
that is transferred depends on how long the two conjugating bacteria remain in contact. In
common laboratory strains of E. coli the transfer of the entire bacterial chromosome takes
about 100 minutes. The transferred DNA can then be integrated into the recipient genome
via homologous recombination. A cell culture that contains in its population cells with
non-integrated F-plasmids usually also contains a few cells that have accidentally
integrated their plasmids. It is these cells that are responsible for the low-frequency
chromosomal gene transfers that occur in such cultures. Some strains of bacteria with an
integrated F-plasmid can be isolated and grown in pure culture. Because such strains
transfer chromosomal genes very efficiently they are called Hfr (high frequency
of recombination). The E. coli genome was originally mapped by interrupted mating
experiments in which various Hfr cells in the process of conjugation were sheared from
recipients after less than 100 minutes (initially using a Waring blender). The genes that
were transferred were then investigated.
Fig. 45. Conjugation – formation of an Hfr cell
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TRANSDUCTION:
Transduction, a process of genetic recombination in bacteria in which genes
from a host cell (a bacterium) are incorporated into the genome of a bacterial
virus (bacteriophage) and then carried to another host cell when the bacteriophage
initiates another cycle of infection. In general transduction, any of the genes of the host
cell may be involved in the process; in special transduction, however, only a few specific
genes are transduced. It has been exploited as a remarkable molecular biological
technique for altering the genetic construction of bacteria, for locating bacterial genes,
and for many other genetic experiments.
Transduction happens through either the lytic cycle or the lysogenic cycle. If the
lysogenic cycle is adopted, the phage chromosome is integrated (by covalent bonds) into
the bacterial chromosome, where it can remain dormant for thousands of generations. If
the lysogen is induced (by UV light for example), the phage genome is excised from the
bacterial chromosome and initiates the lytic cycle, which culminates in lysis of the cell
and the release of phage particles. The lytic cycle leads to the production of new phage
particles which are released by lysis of the host.
Fig. 46. Lytic and lysogenic cyles.
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The packaging of bacteriophage DNA has low fidelity and small pieces of
bacterial DNA, together with the bacteriophage genome, may become packaged into the
bacteriophage genome. At the same time, some phage genes are left behind in the
bacterial chromosome. There are generally three types of recombination events that can
lead to this incorporation of bacterial DNA into the viral DNA, leading to two modes of
recombination.
Generalized Transduction
Generalized transduction is the process by which any bacterial gene may be
transferred to another bacterium via a bacteriophage, and typically carries only bacterial
DNA and no viral DNA. In essence, this is the packaging of bacterial DNA into a viral
envelope. This may occur in two main ways, recombination and headful packaging.
If bacteriophages undertake the lytic cycle of infection upon entering a bacterium,
the virus will take control of the cell’s machinery for use in replicating its own viral
DNA. If by chance bacterial chromosomal DNA is inserted into the viral capsid which is
usually used to encapsulate the viral DNA, the mistake will lead to generalized
transduction.
If the virus replicates using 'headful packaging', it attempts to fill the nucleocapsid with
genetic material. If the viral genome results in spare capacity, viral packaging
mechanisms may incorporate bacterial genetic material into the new virion. The new
virus capsule now loaded with part bacterial DNA continues to infect another bacterial
cell. This bacterial material may become recombined into another bacterium upon
infection.
When the new DNA is inserted into this recipient cell it can fall to one of three fates
1. The DNA will be absorbed by the cell and be recycled for spare parts.
2. If the DNA was originally a plasmid, it will re-circularize inside the new cell and
become a plasmid again.
3. If the new DNA matches with a homologous region of the recipient cell’s
chromosome, it will exchange DNA material similar to the actions
in conjugation.
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This type of recombination is random and the amount recombined depends on the size of
the virus being used.
Fig. 47. Generalized Transduction
Specialized Transduction
Specialized transduction is the process by which genes that are near the bacteriophage
genome may be transferred to another bacterium via a bacteriophage. The genes that get
transferred (donor genes) always depend on where the phage genome is located on the
chromosome. This second type of recombination event which is the result of mistakes in
the transition from a virus' lysogenic to lytic cycle is called specialized transduction, and
non-viral DNA is carried as an insertion/substitution. If a virus incorrectly removes itself
from the bacterial chromosome, bacterial DNA from either end of the phage DNA may
be packaged into the viral capsid. Specialized transduction leads to three possible
outcomes:
1. DNA can be absorbed and recycled for spare parts.
2. The bacterial DNA can match up with a homologous DNA in the recipient cell
and exchange it. The recipient cell now has DNA from both itself and the other
bacterial cell.
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3. DNA can insert itself into the genome of the recipient cell as if still acting like a
virus resulting in a double copy of the bacterial genes.
When the partially encapsulated phage material infects another cell and becomes a
"prophage" (is covalently bonded into the infected cell's chromosome), the partially
coded prophage DNA is called a "heterogenote".
Esther Lederberg, Larry Morse, Herman Kalckar, Michael Yarmolinsky, and Yukinori
Hirota went on to do detailed studies of Galactosemia. Specialized transduction was used
in these studies for gene mapping. At about this time, Esther Lederberg, Julius Adler,
and Enrico Calef were also engaged in similar research involving Maltophilia.
Example of specialized transduction is λ phages in Escherichia coli discovered by Esther
Lederberg as well as Fertility Factor F, also discovered by Esther Lederberg.
Fig. 48. Specialized transduction
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TRANSFORMATION:
Transformation involves the uptake of free or naked DNA released by donor by a
recipient. It was the first example of genetic exchange in bacteria to have been
discovered. This was first demonstrated in an experiment conducted by Griffith in 1928.
The presence of a capsule around some strains of pneumococci gives the colonies a
glistening, smooth (S) appearance while pneumococci lacking capsules have produce
rough (R) colonies. Strains of pneumococci with a capsule (type I) are virulent and can
kill a mouse whereas strains lacking it (type II) are harmless. Griffith found that mice
died when they were injected with a mixture of live non capsulated (R, type II) strains
and heat killed capsulated (S, type I) strains. Neither of these two when injected alone
could kill the mice, only the mixture of two proved fatal. Live S strains with capsule were
isolated from the blood of the animal suggesting that some factor from the dead S cells
converted the R strains into S type. The factor that transformed the other strain was found
to be DNA by Avery, McLeod and McCarty in 1944.
Fig. 49. Evidence of transformation
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Transformation is gene transfer resulting from the uptake by a recipient cell of
naked DNA from a donor cell. Certain bacteria (e.g. Bacillus, Haemophilus, Neisseria,
Pneumococcus) can take up DNA from the environment and the DNA that is taken up
can be incorporated into the recipient's chromosome.
By 1926 the quest to determine the mechanism for genetic inheritance had
reached the molecular level. Previous discoveries by Gregor Mendel, Walter Sutton,
Thomas Hunt Morgan, and numerous other scientists had narrowed the search to the
chromosomes located in the nucleus of most cells. But the question of what molecule was
actually the genetic material had not been answered.
In 1928 Frederick Griffith, in a series of experiments with Diplococcus
pneumonia (bacterium
responsible
for
pneumonia),
witnessed
a
miraculous
transformation. During the course of his experiment, a living organism (bacteria) had
changed in physical form.
The pneumococcus bacterium occurs naturally in two forms with distinctively
different characteristics. The virulent (S-strain) form has a smooth polysaccharide
capsule that is essential for infection. The nonvirulent (R-strain) lacks the polysaccharide
capsule, giving it a rough appearance. Mice injected with S-strain of the pneumococcus
bacteria die from pneumonic infection within a few days, while mice injected with the Rstrain bacteria continue to live. Injection with heat-killed S-strain bacteria also results in
the mice surviving.
Griffith was surprised to find in his experiments that mice injected with a mixture
of heat-killed S-strain and live but nonvirulent R-strain produced lethal results. In fact,
Griffith discovered living forms of the S-strain bacteria in the infected mice. He
hypothesize that the R-strain bacteria had somehow been transformed by the heat-killed
S-strain bacteria. Some "transforming principle", transferred from the heat-killed S-strain,
had enabled the R-strain to synthesize a smooth polysaccharide coat and become virulent.
Oswald Avery, Colin McCleod, and Maclyn McCarty (1934-1944) at the
Rockefeller Institute, building on Griffith's work, showed that only DNA could cause the
transformation. They isolated a cell-free extract from the S-strain bacteria and were able
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to transform living R-strain into a culture containing both S-strain and R-strain cells. The
purified extract contained Griffith's "transforming principle". Through biochemical
testing, they showed it to be deoxyribonucleic acid (DNA).
Fig. 50. Transformation (a) with DNA fragments and (b) with a plasmid
Fig. 51. Mechanism of Transformation
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NPTEL – Biotechnology – Microbiology
REFERENCES:
Text Books:
1. Jeffery C. Pommerville. Alcamo’s Fundamentals of Microbiology (Tenth Edition).
Jones and Bartlett Student edition.
2. Gerard J. Tortora, Berdell R. Funke, Christine L. Case. Pearson - Microbiology: An
Introduction. Benjamin Cummings.
3. J. Krebs, E.S. Goldstein, Stephen T. Kilpatrick. Lewin’s Genes X. Jones and Bartlett
Publishers.
Reference Books:
1. Lansing M. Prescott, John P. Harley and Donald A. Klein. Microbiology. Mc Graw
Hill companies.
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