Chapter I:

Introduction and Concept

Modern molecular biology arose from studies in the separate sciences of bacteriology,
biochemistry, cell biology, and genetics. In many respects, it now weaves together all of these
separate disciplines into a single whole. Nowadays, its influence pervades nearly all of modern
biology and it serves as a unifying approach to the study of many problems in the biological
sciences.
The Central Dogma
The relationship between nucleic acids and proteins is so important to modern biology
that it is called The Central Dogma. The bulk of the course will be taken up with an exploration
of the remarkable properties of biological macromolecules. The Central Dogma itself is
relatively simple, although the chemical mechanisms underlying it are not. The Dogma states:
DNA molecules contain information about how to create proteins; this information is transcribed
into RNA molecules, which, in turn, direct chemical machinery that translates the nucleic acid
message into a protein. The Central Dogma means that the flow of information is one-way,
from DNA to RNA to protein. There are some exceptions to the central dogma! For example,
the AIDS virus, and its relatives in the broader family of retroviruses, is able to translate RNA
into DNA. Hence the term ‘retro’ in retrovirus. However, these kinds of exceptions are quite
rare, and the Central Dogma is about as law-like as any statement in biology ever gets.
Another meaning of the Central Dogma is that nearly* all of the information necessary to
construct and operate a living thing is contained in its DNA. We call the complete complement
of DNA in a living thing its genome.

Chapter II:
Nature of genetic material

Discovery of DNA (Highlights of History)

Friedrich Miescher discovered nucleic acid in 1869. He was a 22-year-old Swiss

Physician. While treating wounded soldiers, he obtained a ready supply of white blood cells
from the pus on the soldier's bandages. When he digested the pus with pepsin and HCl and
extracted the digest with ether, he obtained a preparation of cell nuclei - the first subcellular
fractionation. Then by treating the nuclei with alkali, he obtained a precipitate which he called
nuclein and which because of its acidic properties was later called nucleic acid.
During the 1920s, biochemist P.A. Levene analyzed the components of the DNA
molecule. He found it contained four nitrogenous bases: cytosine, thymine, adenine, and

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guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit
(nucleotide) was composed of a base attached to a sugar and that the phosphate also attached to
the sugar. He (unfortunately) also erroneously concluded that the proportions of bases were
equal and that there was a tetranucleotide that was the repeating structure of the molecule. The
nucleotide, however, remains as the fundamental unit (monomer) of the nucleic acid polymer.
There are four nucleotides: those with cytosine (C), those with guanine (G), those with adenine
(A), and those with thymine (T).
Molecular structure of three nirogenous bases.

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 During the 1920s Frederick Griffith, a medical officer in London whose principal work
was the study of epidemics, studied the difference between a disease-causing strain of the
pneumonia causing bacteria (Streptococcus peumoniae) and a strain that did not cause
pneumonia. The pneumonia-causing strain (the S strain) was surrounded by a capsule. The other
strain (the R strain) did not have a capsule and did not cause pneumonia. Frederick Griffith
(1928) was able to induce a nonpathogenic strain of the bacterium Streptococcus pneumoniae to
become pathogenic. Griffith referred to a ‘transforming factor’ that caused the non-pathogenic
bacteria to become pathogenic.

1. Griffith injected the different strains of bacteria into mice. The S strain killed the
mice; the R strain did not.
2. He further noted that if heat killed S strain was injected into a mouse, it did not
cause pneumonia.
3. When he combined heat-killed S with Live R and injected the mixture into a
mouse (remember neither alone will kill the mouse) that the mouse developed
pneumonia and died.
4. Bacteria recovered from the mouse had a capsule and killed other mice when
injected into them!
5. Further experiments led Griffith to conclude that the Live R had been
transformed into Live S by some "transforming factor".

In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty revisited Griffith's
experiment and concluded the transforming factor was DNA.

The start of a distinct modern molecular biology dates from the discovery of the structure
of DNA by Watson and Crick.
How the structure of DNA was determined?

Jim Watson and Francis Crick proposed a model for the structure of DNA in 1953. They
used molecular models to arrive at their proposed structure. Their model building was
essentially guided by following basic observations:

1. X-ray diffraction studies of DNA fibers.

Watson and Crick depended on X-ray fibre diffraction pictures of DNA -- taken
by Rosalind Franklin -- for their conviction that the structure of DNA was regular and,
ultimately, that it was helical.

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2. Chargaff's Rules.

Erwin Chargaff at Columbia University analyzed the nitrogenous bases in many

different forms of life, concluding that the amount of purines does not always equal the
amount of pyrimidines (as proposed by Levene). DNA had been proven as the genetic
material by the Hershey-Chase experiments, but how DNA served as genes was not yet
certain. The curious feature of his data, which we now know as Chargaff's rules, was
that the amount of adenine nearly always equalled the amount of thymine and the
amount of cytosine nearly always equalled the amount of guanine.

The following table shows some sample data that he collected:

Source mol % of bases Ratios %GC

A G C T A/T G/C
PhiX-174 24.0 23.3 21.5 31.2 0.77 # 1.08 44.8
Maize 26.8 22.8 17.0 * 27.2 0.99 0.98 46.1
Octopus 33.2 17.6 17.6 31.6 1.05 1.00 35.2
Chicken 28.0 22.0 21.6 28.4 0.99 1.02 43.7
Rat 28.6 21.4 20.5 28.4 1.01 1.00 42.9
Human 29.3 20.7 20.0 30.0 0.98 1.04 40.7
(* Plant DNA is an apparent exception to Chargaff's rules; however, plant DNA is often heavily
methylated. As long as the amount of 5-methyl-cytosine is included in the total amount of cytosine, the
rules are obeyed.
# Some bacteriophage DNA's (e.g. PhiX174) do not adhere to Chargaff's rules - these phages have single-
stranded genomes and are therefore not constrained by the requirements of a double-stranded structure. )

Complementarity was a central feature and Chargaff's rules seemed to support the
model. Chargaff's rules were a real key that stood out as a consequence of a double-
helical structure for DNA".

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Nucleic acids are linear, unbranched polymers of nucleotides.
Nucleotides consist of three parts:
 A five-carbon sugar (hence a pentose).
Two kinds are found:
o Deoxyribose, which has a hydrogen atom attached to its #2 carbon atom
(designated 2')
o Ribose, which has a hydroxyl group atom there
Deoxyribose-containing nucleotides, the deoxyribonucleotides, are the monomers of DNA.
Ribose-containing nucleotides, the ribonucleotides, are the monomers of RNA.

 A nitrogen-containing ring structure called a base. The base is attached to the 1' carbon
atom of the pentose.
o In DNA, four different bases are found:
 two purines, called adenine (A) and guanine (G)
 two pyrimidines, called thymine (T) and cytosine (C)
o RNA contains:
 The same purines, adenine (A) and guanine (G).
RNA also uses the pyrimidine cytosine (C), but instead of thymine, it uses
the pyrimidine uracil (U).

The Pyrimidines The Purines

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The combination of a base and a pentose is called a nucleoside.

 One, two, or three phosphate groups

These are attached to the 5' carbon atom of the pentose.

Both DNA and RNA are assembled from nucleoside triphosphates.

In both cases, as each nucleotide is attached, the second and third phosphates are removed.

The practical breakthrough in Watson & Crick's search for the structure of DNA came
when they discovered the base-pairing scheme which allows hydrogen bonds to form between
adenine and thymine and between cytosine and guanine.

The double helix of DNA has these features: (Structure of B-DNA)

 It is an antiparallel double helix.

 It contains two polynucleotide strands wound around each other.

 It is a right-handed helix.

 The backbone of each strand consists of alternating deoxyribose and phosphate groups.

 The phosphate group bonded to the 5' carbon atom of one deoxyribose is covalently bonded
to the 3' carbon of the next.

 The two strands are "antiparallel"; that is, one strand runs 5′ to 3′ while the other runs 3′ to 5′.

 The DNA strands are assembled in the 5′ to 3′ direction and, by convention, we "read" them
the same way.

 The purine or pyrimidine attached to each deoxyribose projects in toward the axis of the
helix.

 Each base forms hydrogen bonds with the one directly opposite it, forming base pairs (also
called nucleotide pairs).
 The base-pairs are perpendicular to the axis of the helix. (Actually, they are very slightly
tilted - at an angle of 4 degrees)

 The axis of the helix passes through the centre of the base pairs.

 3.4 Å separates the planes in which adjacent base pairs are located.

 The double helix makes a complete turn in just over 10 nucleotide pairs, so each turn takes a
little more (35.7 Å to be exact) than the 34 Å shown in the diagram.

 There is an average of 25 hydrogen bonds within each complete turn of the double helix
providing a stability of binding about as strong as what a covalent bond would provide.

 The diameter of the helix is 20 Å.

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 The helix can be virtually any length; when fully stretched, some DNA molecules are as
much as 5 cm (2 inches!) long.

 The path taken by the two backbones forms a major (wider) groove (from "34 A" to the top
of the arrow) and a minor (narrower) groove (the one below).

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 Apart from B form of DNA, two more forms of DNA are known. The comparative account
of these forms is presented in the following chart.

Feature A-Form B-Form Z-Form

Helix type or screw sense Right-handed Right-handed Left-handed
Helix Diameter ~2.6 nm ~2.0 nm ~1.8 nm
Base pairs per turn of helix 11 10.4 12
Helix pitch or rise per turn of
2.53 nm ~3.54 nm 4.56 nm
the helix
36 degrees
Helical twist or rotation per
33 degrees (actual 27-42 with ~34.6 60 degrees
successive bp
average)
Tilt of basepair from helix
19 degrees 6 degrees (1 book) 9 degrees
axis
narrow and very
Major groove Wide and quite deep Flat
deep
Very broad and Very narrow and
Minor groove Narrow and quite deep
shallow deep
C2'-endo for pyr
Sugar pucker C3'-endo C2'-endo
C3'-endo for pur
Anti for pyr
Glycosidic bond Anti Anti
Syn for pur
Overall shapes of double
Broadest Intermediate Narrowest
helix