Semester-III: Paper-II. Unit-III: Molecular Biology. Dr. Ajita Kumar.

PAPER III: Unit III : Molecular Biology- SYLLABUS

● DNA replication :
○ Replication- Prokaryotic and Eukaryotic
○ Protein Synthesis:
■ Central dogma of Protein synthesis
■ Transcription:
● The transcription process in prokaryotes and eukaryotes,
● mRNA synthesis,
● mRNA processing,
● Adenylation & Capping

Molecular Biology
INTRODUCTION
➢ This branch of biology was established in the 1930s, the term was coined by Warren
Weaver in 1938, then director of Natural Sciences for the Rockefeller Foundation.
➢ Since the late 1950s and early 1960s, molecular biologists have learned to
characterize, isolate, and manipulate the molecular components of cells and organisms.
➢ From the early 2000s, the study of gene structure and function, molecular genetics, has
been among the most prominent sub-field of molecular biology.
➢ Clinical research and medical therapies arising from molecular biology are partly covered
under gene therapy.
➢ Molecular biology also plays an important role in understanding formations, actions,
regulations of various parts of cells which can be used efficiently for targeting new drugs,
diagnosis of disease and physiology of the Cell.
➢ Molecular Biology is thus, the study of the structure and function of the
macromolecules found in living things. It deals with the molecular basis of biological
activity and chiefly concerns understanding the interactions between the various systems
of a cell, including the interactions between the different types of DNA, RNA and protein
biosynthesis as well as learning how these interactions are regulated.
➢ Nucleic acids are the macromolecules that store (Genes in the DNA) and
transfer(Replication) hereditary information. It also provides instructions for making
proteins(Transcription and translation).
DNA REPLICATION
➢ Two well established and the only functions of the DNA are categorised as
autocatalytic function and heterocatalytic function.
➢ Autocatalytic function of the DNA is to replicate itself.
○ With Mendel’s experiments on genetics it became clear that heredity had a
biochemical basis for inheritance.

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 Semester-III: Paper-II. Unit-III: Molecular Biology. Dr. Ajita Kumar.
○ The experiments also demonstrated that nucleic acids were transmitted from
one generation to the next.
○ Therefore the DNA molecule needs to be replicated, that is to produce a copy of
its own in order to be transmitted.
○ Life begins from a single cell that is the zygote formed after fertilization. Then, all
the cells and tissue types are produced by cell division.
○ The maintenance of normal functions in an adult requires constant mitosis.
➢ REPLICATION AND HEREDITY
○ It is established that DNA is the basis of heredity in all living organisms
○ DNA of each organism is distinguished by a specific sequence of the
nitrogenous bases appearing within the molecule.
○ The sequence of the nitrogenous bases constitute the structure of the genes.
○ The genes control the genetic characters.
○ Thus the DNA replicates producing exactly the same copies of itself to transmit
the characters to the next generation identical to the ones present in the parents.
○ Also errors in replication may lead to genetic disorders.
DNA REPLICATION MODEL
During cell division, each DNA molecule has to be perfectly copied to ensure identical
DNA molecules move to each of the two daughter cells.
The double-stranded structure of DNA suggested that the two strands might separate
during replication. Then each of the separated strands serves as a template from which
the new complementary strand for each is copied, generating two double-stranded
molecules from one.

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 Semester-III: Paper-II. Unit-III: Molecular Biology. Dr. Ajita Kumar.
There were three possible models of replication from such a scheme: conservative,
semi-conservative, and dispersive.
● Conservative Models of Replication-
○ In conservative replication, the two original DNA strands, known as the
parental strands, would re-base pair with each other after being used as
templates to synthesize new strands; and
○ The two newly-synthesized strands, known as the daughter strands, would also
base-pair with each other; one of the two DNA molecules after replication would
be “all-old” and the other would be “all-new”.
● Semi-conservative Models of Replication-
○ In semi-conservative replication, each
of the two parental DNA strands would
act as a template for new DNA strands to
be synthesized, but
○ After replication, each parental DNA
strand would base-pair with the
complementary newly-synthesized
strand, and
○ Both double-stranded DNAs would
include one parental or “old” strand
and one daughter or “new” strand.
● Dispersive Models of Replication-
○ In dispersive replication, after replication
both copies of the new DNAs would
somehow have alternating segments of
parental DNA and newly-synthesized
DNA on each of their two strands.
Meselson and Stahl Experiment- Steps
● To determine which model of replication was accurate, an experiment was performed in
1958 by two researchers: Matthew Meselson and Franklin Stahl.
● Meselson and Stahl, using E. coli DNA made with two nitrogen isotopes (14N and 15N)
and density gradient centrifugation, determined that DNA replicated via the
semi-conservative method of replication.
● They grew E. coli for several generations in a medium containing a “heavy” isotope of
nitrogen (15N).
● The isotope got incorporated into nitrogenous bases and, eventually, into the DNA.
● This E. coli culture was then shifted into a medium containing the common “light”
isotope of nitrogen (14N) and allowed to grow for one generation.
● After one generation some cells were harvested and the DNA was isolated.
● Remaining cells were allowed to grow further in 14N and then the DNA was isolated.
● This was repeated, allowing the cells to grow further after every isolation.
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● Isolated DNA was centrifuged in density gradient ultracentrifugation
● During the density gradient ultracentrifugation, the DNA was loaded into a cesium
chloride gradient and spun at high speeds of 50,000 to 60,000 rpm.
● In the ultracentrifuge tube, the cesium chloride salt created a density gradient, with the
cesium chloride solution being more dense the farther down the tube.
● Under these circumstances, during the spin the DNA was pulled down the
ultracentrifuge tube by centrifugal force until it arrived at the spot in the salt gradient
where the DNA molecules’ density matched that of the surrounding salt solution.
● At the point, the molecules stopped sedimenting and formed a stable band.
● So DNA grown in 15N had a higher density with a heavier isotope of nitrogen
incorporated into its nitrogenous bases.
Meselson and Stahl Experiment- Observations
● E. coli were first grown in heavy nitrogen (15N) then in lighter nitrogen (14N.)
● After one generation of growth in 14N, the DNA molecules produced only a single band
which was intermediate in position i.e between DNA of cells grown exclusively in 15N
and DNA of cells grown exclusively in 14N.
● The single band actually seen indicated that all the DNA molecules contained equal
amounts of both 15N and 14N.
● This suggested either a semi-conservative or dispersive mode of replication.
● Conservative replication would have resulted in two bands; one representing the
parental DNA still with exclusively 15N in its nitrogenous bases and the other
representing the daughter DNA with exclusively 14N in its nitrogenous bases.
● The DNA harvested from cells grown for two generations in 14N formed two bands: one
DNA band was at the intermediate position between 15N and 14N and the other
corresponded to the band of exclusively 14N DNA.
● These results could only be explained if DNA replicates in a semi-conservative manner.
● Dispersive replication would have resulted in exclusively a single band in each new
generation, with the band slowly moving up closer to the height of the 14N DNA band.
● Therefore, dispersive replication could also be ruled out.
● DNA grown in 15N (red band) is heavier than DNA grown in 14N (orange band) and
sediments to a lower level in the cesium chloride density gradient in an ultracentrifuge.
● When DNA grown in 15N is switched to media containing 14N, after one round of cell
division the DNA sediments halfway between the 15N and 14N levels, indicating that it
now contains fifty percent 14N and fifty percent 15N.
● In subsequent cell divisions, an increasing amount of DNA contains 14N only. These
data support the semi-conservative replication model.
● DNA from cells grown exclusively in 15N produced a lower band than DNA from cells
grown exclusively in 14N.
Meselson and Stahl Experiment- Conclusion
1. Thus during DNA replication, each of the two strands that make up the
double helix serves as a template from which new strands are synthesized.
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 Semester-III: Paper-II. Unit-III: Molecular Biology. Dr. Ajita Kumar.
2. The new strand will be complementary to the parental or “old” strand and the new strand
will remain base-paired to the old strand.
3. So each “daughter” DNA actually consists of one “old” DNA strand and one
newly-synthesized strand.
4. When two daughter DNA copies are formed, they have the identical sequences to one
another and identical sequences to the original parental DNA.
5. The two daughter DNAs are divided equally into the two daughter cells.
6. Producing daughter cells that are genetically identical to one another and genetically
identical to the parent cell.

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