Module 1: Central Dogmas

→ Central Dogma of Molecular Biology

→ DNA as genetic material
→ Structure of DNA and RNA

CENTRAL DOGMA

▪ The concept of central dogma was first proposed by Francis Crick in 1958.
▪ The ‘Central Dogma’ is the process by which the instructions in DNA are converted into a functional
product.
▪ The central dogma of molecular biology explains the flow of genetic information, from DNA to RNA,
to make a functional product, a protein.
▪ The central dogma suggests that DNA contains the information needed to make all of our proteins,
and that RNA is a messenger that carries this information to the ribosome.
▪ The process by which the DNA instructions are converted into the functional product is called gene
expression.
▪ Gene expression has two key stages - Transcription and Translation
▪ The central dogma states that the pattern of information that occurs most frequently in our cells is:
○ From existing DNA to make new DNA (DNA replication)
○ From DNA to make new RNA (transcription)
○ From RNA to make new proteins (translation).
DNA REPLICATION:

DNA REPLICATION:
▪ It is the replication of genetic material.
▪ The strand of DNA are separated and results the formation of two new strand of DNA with help of
several enzymes.
▪ In both newly synthesized DNA, one strand is new and one strand is old (SEMI-CONSERVATIVE)

TRANSCRIPTION:
▪ In transcription, the information in the DNA of every cell is converted into small, portable RNA
messages.
▪ The process does not require primer but involve multiple RNA polymerase.

Reverse transcription is the transfer of information from RNA to make new DNA, this occurs in the case of
retroviruses, such as HIV. It is the process by which the genetic information from RNA is assembled into new
DNA.

TRANSLATION:
▪ During translation, these messages travel from where the DNA is in the cell nucleus to the ribosomes .
▪ The ribosomes serve as factories in the cell where the information is ‘translated’ from a code into the
functional product (protein)

DNA AS GENETIC MATERIAL

The genetic material must have three key characteristics:

1. It must contain, in a stable form, the information about an organism’s cell structure, function,
development, and reproduction.
2. It must replicate accurately, so that progeny cells have the same genetic information as the parental
cell.
3. It must be capable of change. Without change, organ  isms would be incapable of varia on and
adaptation, and evolution could not occur.
Friedrich Miescher, a Swiss biochemist is credited for the discovery of nucleic acid in 1869. He isolated a
substance from white blood cells of pus in used bandages during the Crimean War.
At first he believed the substance to be protein; but chemical tests indicated that it contained carbon,
hydrogen, oxygen, nitrogen, and phosphorus, the last of which was not known to be a component of
proteins.
Searching for the same substance in other sources, Miescher found it in the nucleus of all the samples he
studied—and, therefore, he called it nuclein. However, its function and location in the cell remained
unknown.

In early 1900s, experiments showed that chromosomes that are made of protein and nucleic acid(DNA and
RNA) were the carrier of genetic information from one generation to the next.
Scientists reasoned that protein must be the genetic material and they have a great capacity for storing
information because they were composed of 20 different amino acids.
DNA, with its four nucleotides, was thought to be too simple a molecule to account for the variation found in
living organisms.
However, beginning in the late 1920s, a series of experiments led to the definitive identification of DNA as genetic
material.

Griffith’s Transformation Experiment:

 In 1928, Griffith Frederick, a British bacteriologist was working on Streptococcus pneumoniae, a pneumonia
causing bacteria.
 Griffith used two strains of bacteria
1. S strain, which produces smooth, shiny colonies and is virulent
□ The virulence of the S strain is due to the presence of a polysaccharide coat, a capsule
surrounding each cell. The coat is also the reason for the smooth, shiny appearance of S
colonies.
2. R strain, which produces rough colonies and is nonvirulent
□ The R strain is genetically identical except that it carries a mutation that prevents it from
making the polysaccharide coat and, hence, alters the virulence state of the bacterium.

Griffith worked with IIS and IIIS strains, which have type II and type III coats, respectively.
Occasionally, S-type cells mutate into R-type cells, and R-type cells mutate into S-type cells.
The mutations are type-specific, meaning that, if a IIS cell mutates into an R cell, then that R cell can mutate
back only into a IIS cell, not a IIIS cell

 Griffith injected mice with different strains of the bacterium and observed their effects on the mice .
1. When mice were injected with IIR bacteria (R bacteria derived by mutation from IIS bacteria),
the mice lived.
2. When mice were injected with living IIIS bacteria, the mice died, and living IIIS bacteria was
isolated from the blood.
3. When mice were injected with heat killed IIIS bacteria, the mice lived
4. When mice were injected with a mixture of living IIR bacteria and heat-killed IIIS bacteria. The
mice died, and living IIIS bacteria were present in the blood. (These bacteria could not have
arisen by mutation of the IIR bacteria, because mutation would have produced IIS bacteria)

 Griffith concluded that some IIR bacteria had somehow been transformed into smooth, virulent IIIS bacteria
by interaction with the dead IIIS bacteria.
 Genetic material from the dead IIIS bacteria had been added to the genetic material in the living IIR bacteria.

" Transformation is the process of picking up the naked genetic material from the surrounding or dead bacteria
(donor) by the living bacteria (recipient) to form a recombinant."
Avery’s Transformation Experiment:

 In the 1930s and 1940s, American biologist Oswald T.Avery, along with his colleagues Colin M. MacLeod
and Maclyn McCarty, tried to identify Griffith’s transforming principle by studying the transformation of R-
type bacteria to S-type bacteria in the test tube.

 They lysed (broke open) IIIS cells with a detergent and used a centrifuge to separate the cellular
components and removed lipids and carbohydrate from the solution.
 They incubated the extract with a culture of living IIR bacteria and then plated cells on a culture medium in
a Petri dish.
 Colonies of IIIS bacteria grew on the plate, showing that the extract contained, the transforming principle,
the genetic material from IIIS bacteria capable of transforming IIR bacteria into IIIS bacteria.
 Avery and his colleagues knew that one of the macromolecular components in the extract— proteins,
RNA, or DNA must be the transforming principle.

 They treated the extract with enzymes that could degrade these macromolecules.
1. When treated with protease (protein degraded), IIIS bacteria appeared, transformation
occurred.
2. When treated with RNAase (RNA degraded), IIIS bacteria appeared, transformation occurred.
3. When treated with DNAase (DNA degraded), IIIS bacteria did not appear, transformation did
not occur.

 They found that the extract failed to bring about transformation only when DNA had been degraded,
despite the presence of all other remaining macromolecules in the extract.
 By contrast, any enzyme treatment that did not lead to digestion of the DNA did not eliminate the
transforming principle.
 These results showed that DNA, and DNA alone, must have been the transforming principle (the genetic
material).

Despite this finding, the scientific community was reluctant to accept the role of DNA as a genetic material
It was only 8 years later, when Hershey and Chase conducted their experiment, that the concept gained traction
Hershey and Chase’s Bacteriophage Experiment:

 In 1953, Alfred D. Hershey and Martha Chase published a paper that provided more evidence that DNA
was the genetic material.
 In their experiments, Hershey and Chase analyzed what happened when 𝑇 phages infect bacteria (E.Coli).
 By the 1950s, scientists had evidence for how phages infected bacteria.
→ when phages infect a host bacterium, the phages first attach themselves to the outside of the
bacterium.
→ Then, a piece of the phage enters the bacterium and subsequently replicates itself inside the
cell.
→ After many replications, the phage causes the bacterium to lyse, or burst, thereby killing the
host bacteria. Scientists classified the replicating piece as genetic material.
Scientists also found that phages contained two classes of biological molecules: DNA and protein.

 To perform their experiments, Hershey and Chase utilized a technique called radioactive isotope labeling.
 There were two batches of 𝑇 : one labelled with radioactive Phosphorous (𝑃 ) and other with labelled
radioactive Sulphur (𝑆 ).
→ They used these isotopes because DNA contains phosphorus but no sulfur, and protein
contains sulfur but no phosphorus.
→ DNA was labelled with 𝑃 and Protein was labelled with 𝑆 .

 Next, they infected the E.Coli with one or the other batch of 𝑇 phage and then the cells were blended in
kitchen blender.
→ When the infecting phage was 𝑃32 -labeled, most of the radioactivity was found within the
bacteria soon after infection. Very little was found in the phage ghosts
→ When the infecting phage was 𝑆 -labeled, most of the radioactivity was found in the phage
ghost. Very little to no radioactivity was found in the E.Coli cell.

 Hershey and Chase concluded that DNA is the genetic

material as it enters the E.Coli evident by the presence
of radioactive 𝑃32 and not radioactive 𝑆 35 in E.coli.

 Alfred Hershey shared the 1969 Nobel Prize in

Physiology or Medicine for his “discoveries concerning
the genetic structure of viruses.”

Keynote
A series of experiments proved that the genetic material
consists of one of two types of nucleic acids: DNA or RNA.
Of the two, DNA is the genetic material of all living
organisms (Prokaryotes and Eukaryotes) and of some
viruses, and RNA is the genetic material of the remaining
viruses.
STRUCTURE OF DNA AND RNA

DNA and RNA are nucleic acids.

Nucleic acids are polymers of nucleotides. A nucleotide consists of a nitrogenous base (purine or pyrimidine), a
pentose sugar, and one or more phosphate groups.

Nitrogenous bases:
The nitrogenous bases found in nucleotides are aromatic heterocyclic
compounds.
The bases are of two types—purines and pyrimidines

Purines- Nine-membered, double-ringed structures. There are two purines:

Adenine (A) and Guanine (G)

Pyrimidines- Six-membered, single-ringed structures. There are three

different pyrimidines: Thymine (T), Uracil (U), Cytosine (C).

Pentose Sugar:
In DNA, the pentose sugar is deoxyribose, and in RNA it is ribose (Figure 7).
The two sugars differ by the chemical groups attached to the carbon: a
hydrogen atom (H) in deoxyribose and a hydroxyl group (OH) in ribose.

 The nitrogenous bases are covalently (N-glycosidic bonds) attached to the 1' carbon of the pentose sugar.
The purine bases are bonded at the 9 nitrogen, and the pyrimidines bond at the1 nitrogen. The
combination of a sugar and a base is called a nucleoside.
 Addition of phosphate group (𝑃𝑂 ) to a nucleoside yields nucleotide. The phosphate group is attached
to the 5' carbon of pentose sugar by phosphodiester bond.
 To form polynucleotides , nucleotides are linked together by a covalent bond between the phosphate
group of one nucleotide and the 3' carbon of the sugar of another nucleotide. These 5' -to-3' phosphate
linkages are called phosphodiester bond.

STRUCTURE OF DNA:

Two lines of investigation helped in determining the structure of DNA:

1. X-Ray Crystallography- Maurice Wilkins and Rosalind Franklin obtained a very fine X-ray diffraction
pictures of DNA. They concluded that DNA is a helical structure with two distinctive regularities of 0.34
nm and 3.4 nm along the axis of molecule.
2. Chargaff's Rules- Erwin Chargaff performed some base composition studies on different organism and
out forward certain generalizations for ds-DNA.
 Purines and Pyrimidines occur in equal amount.
 The amount of adenine ( A) was equal to that of thymine ( T), and the amount of guanine (G) was
equal to that of cytosine ( C).
 𝐴+𝐺
 ⎯⎯⎯⎯⎯= 1. This values was constant for all species.
𝑇+𝐶
𝐴+𝑇
 ⎯⎯⎯⎯⎯ (𝐵𝑎𝑠𝑒 𝑅𝑎𝑡𝑖𝑜) 𝑤𝑎𝑠 𝑠𝑝𝑒𝑐𝑖𝑒𝑠 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐.
𝐺+𝐶
 Base ratio of prokaryotes is less than 1 (eg- E.coli=0.92) and for eukaryotes it is always more
than one (eg- human=1.67)

WATSON AND CRICK MODEL OF DNA:

James Watson and Francis Crick used the data obtained from Chargaff's base composition studies and X-Ray
crystallography by Rosalind and Franklin and proposed the double helical model for structure of DNA in 1953.

Salient features of double helix structure of DNA are:

1. The DNA consists of two polynucleotide chains. The double chain of DNA is helically twisted in a right-
handed fashion.
2. The two chains of DNA are antiparallel; i.e. the two strands are oriented in opposite directions with one
strand oriented in 5'-3' direction and the other oriented in 3'-5' direction.
3. The sugar–phosphate backbones are on the outsides of the double helix, with the bases oriented toward
the central axis . The bases of both chains are flat structures oriented perpendicularly to the long axis of
the DNA so that they are stacked like pennies on top of one another, following the twist of the helix.
4. The bases in each of the two polynucleotide chains are bonded together by hydrogen bonds. Adenine
forms 2 H-bonds with Thymine and Guanine forms 3 H-bonds with Cytosine. The specific A–T and G–C
pairs are called complementary base pairs.
For instance, if one chain has the sequence 5'-TATTCCGA-3', then the opposite, antiparallel chain must
bear the sequence 3'-ATAAGGCT-5'.
5. Each turn of double helix or the pitch of the helix is 3.4 nm and the distance between two consecutive
base pairs is 0.34 nm. Therefore, there are 10 base pairs (bp) per turn.
6. Because of the way the bases bond with each other, the two sugar–phosphate backbones of the double
helix are not equally spaced from one another along the helical axis. This unequal spacing results in
grooves of unequal size between the backbones; one groove is called the major (wider) groove, the other
the minor (narrower) groove. The space of major groove and minor groove is 2.2 nm and 1.2 nm.

For their “discoveries concerning the molecular

structure of nucleic acids and its significance for
information transfer in living material,” the 1962
Nobel Prize in Physiology or Medicine was awarded to
Francis Crick, James Watson, and Maurice Wilkins.
What was Rosalind Franklin’s contribution to the
discovery? This has been the subject of debate, and we
will never know whether she would have shared the
prize. She died in 1962, and Nobel Prizes are never
awarded posthumously.
DIFFERENT FORMS OF DNA:
Researchers have now shown that DNA can exist in several different forms—most notably, the A-DNA, B-DNA,
C-DNA and Z-DNA form.

Helix Base Pair Rotation Hight/Bp Diameter Condition

A 11bp Righthanded 2.3 A0 25.5 A0 75% relative humidity Na+, K+, Cs+
B 10bp Righthanded 3.4 A0 23.5 A0 92% relative humidity
low ionic strength
C 9.33bp Righthanded 3.3 A0 23.7 A0 66% relative humidity and Lithium and
magnesium ions
Z 12bp Left handed 5.7 A0 18.4 A0 Very low salt concentration

STRUCTURE OF RNA:

RNA is a polymer of ribonucleotides held together by 3c,5c-phosphodiester bridges. Although RNA has certain
similarities with DNA structure, they have specific differences
1. Pentose: The sugar in RNA is ribose in contrast to deoxyribose in DNA.
2. Pyrimidine: RNA contains the pyrimidine uracil in place of thymine (in DNA).
3. Single strand: RNA is usually a single stranded polynucleotide. However, this strand may fold at certain
places to give a double stranded structure, if complementary base pairs are in close proximity.
4. Chargaff’s rule—not obeyed: Due to the single-stranded nature, there is no specific relation between
purine and pyrimidine contents. Thus the guanine content is not equal to cytosine (as is the case in DNA).
5. Susceptibility to alkali hydrolysis: Alkali can hydrolyze RNA to 2',3'-cyclic diesters. This is possible due to
the presence of a hydroxyl group at 2' position. DNA cannot be subjected to alkali hydrolysis due to lack
of this group.