Microbial Molecular Biology and Genetics

91 Pages·2014·3.08 MB·English

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.
• 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
• Watson and Crick summarized the above findings and postulated their model of
DNA structure.
• A, B, Z DNA
• RNA structure
Griffith Experiment
• The bacteria in the experiment had two different strains: R strain (harmless) and S
strain (harmful).
• Live R strain had no effect on the mice, while live S strain killed the mice.
• Heat-killed S strain failed to kill the mice.
• When live R strain (harmless) was mixed with heat-killed S strain (now
harmless), and the injected mice died.
• 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 heat-
killed cells, although they were not living, still passed their hereditary material to the
living R cells somehow.
Avery-MacLeod-McCarty Experiment
• Extracted components from heat-killed S bacteria.
• After each extraction, S cells were mixed with R bacteria.
• R bacteria transformed each time until DNA was extracted from S cells.
• 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)

• Labeled bacteriophage DNA with radioactive phosphorus
• After bacteriophage infected bacteria, phosphorus was found in bacteria
• Labeled protein coat with radioactive sulfur
• After bacteriophage infected bacteria, no sulfur was found in bacteria
• 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 Bases
Adenine Guanine Cytosine Uracyl

(A) (G) (C) (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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