CHEMISTRY-VIII NOTES PREPARED BY Dr.

DHONDIBA VISHWANATH SURYAWANSHI, GFGC KR PURAM BENGALURU-36

Molecular biology 4 hours Max. Marks: 10

Central dogma of molecular biology, semi conservative replication and mechanism
of DNA replication, Genetic code: general features. Definition of transcription and
translation (mechanism of translation). DNA finger printing- Definition and its
applications.

Molecular biology: The branch of biochemistry which deals with the study of
relationship between the structure and function of biological molecules specially
DNA, RNA and proteins, and how these relationships contribute to the operation
and control of biochemical processes is called molecular biology.
Informational molecules are those whose sequence of subunits reflects or specifies
the sequence of subunits in some other molecule. Example- The sequence of
nucleotides in DNA specifies the sequence of RNA synthesized off it. This RNA in
turn specifies the sequence of amino acids in the protein synthesized from it.
The central dogma of molecular biology: The Watson –Crick model of DNA
had an inbuilt precise mechanism of replication of DNA. It led to the formulation
of the so called central dogma of molecular biology, which is genetic information
flows from DNA to RNA to protein. The central dogma therefore defines three
steps in transfer of genetic information.
1) Replication: This is the copying of parent DNA to form two (daughter)
DNA molecules identical to the parent DNA
2) Transcription: This is the process by which the genetic information present
in one strand of DNA (in the form of its base sequence) is used to synthesize
a complementary sequence of bases in mRNA.
3) Translation: This is the process in which the genetic information encoded
in an mRNA molecule is used to specify the sequence of amino acids during
synthesis of proteins.
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Reverse transcription and RNA replication occur in retroviruses. In

Reverse transcription, the base sequence of RNA specifies the synthesis of
complementary sequence of DNA and performed by an enzyme called
reverse transcription.
Replication of DNA (DNA Synthesis): In eukaryotic organisms, DNA is
present in the form of chromosomes in the nuclei of cells. In addition, they
also contain circular DNA in mitochondria. Plants also contain circular DNA
in their chloroplasts. Prokaryotic cells lack proper nuclei, but have a single
chromosome. They may also contain nonchromosomal DNA in the form of
plasmids.
The fertilized eggs of an organism each contain DNA which encodes the
complete blueprint of the organism, which directs its development. In multi-
cellular organisms, this may involve the production and growth of trillions
of cells. Clearly, DNA replication must take place before every cell division.
Semi conservative replications of DNA: The basic process by which DNA
replicates is similar in all living organisms, and is called semi conservative
replication. In this process, after separation of the two strands, each strand
serves as a template for the synthesis of complementary strand. Thus, each
of the two new DNA molecules contains one old strand and one newly
synthesized strand.

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Mechanism of DNA replication: Prokaryotic DNA replication involves

many proteins. These are collectively referred to as the replisome. The
process has several steps.
1) DNA uncoiling: The E.coli chromosome is a large circular duplex (double
strand) of 4.6 x 103 kilo base (kb) pairs(1kb = 1000 complementary base
pairs). Replication always begins at a precise point of origin, designated as
ori C as shown in figure. This is recognized by a protein (DNA protein). The

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two parental DNA strands separate, forming two Vs where active synthesis
takes place. These are called replication forks, and progressively move away
from the origin as replication occurs i.e. replication is bi-directional. DNA
synthesis occurs simultaneously at both replication forks.

Unwinding of the helix is done by enzymes called helicases, which are

assigned by various topisomaerases, especially DNA gyrase (helicases and DNA
gyrase require ATP). Once unwind, the single DNA strands are kept separate by
many molecules of single stranded DNA binding protein (SSB). SSB probably also
protects the single stranded DNA against attack by nucleases as shown in figure.

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2) Synthesis of RNA primers: The enzyme that synthesize DNA is DNA

polymerase III. However, this cannot initiate DNA synthesis on its own.
DNA polymerase III requires a short RNA primer (about 5-10 nucleotides
long), to which it can add on DNA complementary to the parental DNA
strand. The RNA primers are synthesized by an RNA polymerse called
primase. In a 5’ to 3’ direction. DNA polymerase III then adds on DNA,
attached to the 3’ end of the RNA primer as shown in above figure.
3) DNA synthesis at replication fork: DNA complementary to both parental
strands are copied at the same time in the direction of the replication fork.
All DNA plolymerases synthesise DNA only in the 5’ to 3’ direction (of the
new strand). Thus, on the parent strand with 3’ to 5’ orientation, new DNA
is synthesized as a continuous piece in the 5’ 3’ direction (i.e. in the same
direction as fork movement). This DNA is called the leading strand. On the
other template strand (with 5’ 3’ orientation), DNA polymerase III
synthesizes a short piece of complementary DNA in the 5’ to3’ direction as
and when the parental DNA unwinds. These short fragments of new DNA
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(About 100 -2000 nucleotides long) are synthesized in the direction opposite
that of fork movement, and are called Okazaki fragments. Each Okazaki
fragment contains new DNA attached to the short RNA primer at the 5’ end.
This new DNA stand, made discontinuously, is called the lagging strand, as
it is synthesized slower than the leading strand. As shown in figure.

4) Removal of RNA primers: The single RNA primer at the 5’ end of the
leading strand and the numerous RNA primers at the 5’ ends of each
Okazaki fragment are removed by DNA polymerase I. This simultaneously
replaces the RNA primer with DNA complementary to the parent strands
thereby extending each Okazaki fragment on the lagging strand. As shown
in figure

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5) Joining of DNA fragments: The DNA fragments produced by all combined

action of DNA polymerase III and DNA polymerase I are then joined
together by DNA ligase. Replication ends when the two replication forks
meet on the opposite side of the circular chromosomes.

The whole process of DNA replication is shown below.

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Functions of bacterial (prokaryotic) DNA polymerase

Polymerase Functions
DNA polymerase I (Pol I) Removal of RNA primer
replacement of RNA primer with
DNA, DNA repair
DNA polymerase II (Pol II) DNA repair
DNA Polymerase III (Pol III) DNA synthesis (replication)

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CHEMISTRY-VIII NOTES PREPARED BY Dr. DHONDIBA VISHWANATH SURYAWANSHI, GFGC KR PURAM BENGALURU-36

The action of DNA polymerases: All three DNA polymerases from E. coli
synthesize DNA in the 5’ 3’ direction. This DNA synthesis is template
directed, with the new DNA being complementary to the parent strand (i.e. adenine
opposite thymine and vice versa, guanine opposite cytosine and vice versa)
They need a primer with a free 3’ – OH group and also require all four
deoxyribonucleotide triphosphate (i.e. dADP, dGTP, dCTP and dTTP), in addition
to Mg2+ ions. All contain Zn2+ as cofactor and release pyrophosphate (PPi) on
adding each nucleotide to the DNA chain. The stepwise reaction is

Overall:

Genetic code: The genetic code is the set of rules used by

living cells to translate information encoded within genetic material
(DNA or mRNA sequences of nucleotide triplets, or codons) into proteins.
Translation is accomplished by the ribosome, which links proteinogenic amino
acids in an order specified by messenger RNA (mRNA), using transfer
RNA (tRNA) molecules to carry amino acids and to read the mRNA
three nucleotides at a time. The genetic code is highly similar among all organisms
Or
Genetic code, the sequence of nucleotides in deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA) that determines the amino acid sequence of proteins.

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Though the linear sequence of nucleotides in DNA contains the information

for protein sequences, proteins are not made directly from DNA. Instead,
a messenger RNA (mRNA) molecule is synthesized from the DNA and directs the
formation of the protein. RNA is composed of four nucleotides: adenine (A),
guanine (G), cytosine (C), and uracil (U). Three adjacent nucleotides constitute a
unit known as the codon, which codes for an amino acid. For example, the
sequence AUG is a codon that specifies the amino acid methionine. There are 64
possible codons, three of which do not code for amino acids but indicate the end of
a protein. The remaining 61 codons specify the 20 amino acids that make up
proteins. The AUG codon, in addition to coding for methionine, is found at the
beginning of every mRNA and indicates the start of a protein. Methionine
and tryptophan are the only two amino acids that are coded for by just a single
codon (AUG and UGG, respectively). The other 18 amino acids are coded for by
two to six codons. Because most of the 20 amino acids are coded for by more than
one codon, the code is called degenerate.

General features (Characteristics) of the genetic code: The general features of

genetic codes are given below
1) Triplet nature: A triplet code could make a genetic code for 64 different
combinations (4 X 4 X 4) genetic code and provide plenty of information in
the DNA molecule to specify the placement of all 20 amino acids. When
experiments were performed to crack the genetic code it was found to be a
code that was triplet. These three letter codes of nucleotides (AUG, AAA,
etc.) are called codons.
2) Degeneracy: The code is degenerate which means that the same amino acid
is coded by more than one base triplet. For example, the three amino acids
arginine, alanine and leucine each have six synonymous codons.

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3) Non-overlapping: The genetic code is nonoverlapping, i.e.,the adjacent

codons do not overlap. A nonoverlapping code means that the same letter is
not used for two different codons. In other words, no single base can take
part in the formation of more than one codon.
4) Commaless: There is no signal to indicate the end of one codon and the
beginning of the next. The genetic code is commaless (or comma-free).
5) Non-ambiguity: A particular codon will always code for the same amino
acid. While the same amino acid can be coded by more than one codon (the
code is degenerate), the same codon shall not code for two or more different
amino acids (non-ambiguous).
6) Universality: Although the code is based on work conducted on the
bacterium Escherichia coli but it is valid for other organisms. This important
characteristic of the genetic code is called its universality. It means that the
same sequences of 3 bases encode the same amino acids in all life forms
from simple microorganisms to complex, multicelled organisms such as
human beings.
7) Polarity: The genetic code has polarity, that is, the code is always read in a
fixed direction, i.e., in the 5′ → 3′ direction.
8) Chain Initiation Codons: The triplets AUG and GUG play double roles
in E. coli. When they occur in between the two ends of a cistron
(intermediate position), they code for the amino acids methionine and valine,
respectively in an intermediate position in the protein molecule.
9) Chain Termination Codons: The 3 triplets UAA, UAG, UGA do not code
for any amino acid. They were originally described as non-sense codons, as
against the remaining 61 codons, which are termed as sense codons
Transcription (RNA synthesis): Transcription is the synthesis of RNA from a
DNA template. It is the first step in the transfer of the genetic message from DNA,
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the second step being translation. Transcription is catalyzed by enzymes called

RNA polymerases. These copy a DNA templet, which temporarily unfolds. An
RNA molecule is synthesized in the 5’ 3’ direction. They can initiate the
synthesis of new strands and do not require a primer like DNA polymerases do.
The RNA molecule synthesized will have a base sequence complementary to the
parent DNA strand being copied. Only one strand of the parent DNA duplex is
transcript. This strand is called the antisense strand and the opposite strand of DNA
not copied is called the sense strand.
The process requires the four ribonucleoside 5’-triphosphates as precursors (ATP,
GTP, CTP and UTP). The overall reaction is.

Gene transcription by prokaryotic RNA polymerase takes place in three phases-

initiation, elongation and termination.
1) Initiation: RNA polymerase recognizes a specific signal for
transcription on the DNA, upstream form (i.e. ahead of the 5’ end of)
the gene to be copied. This site is called a promoter site, and is rich in
adenine and thymine. Since only two hydrogen bonds hold together A
and T (is opposed to three which hold together G and C), the parental
DNA strands can be separated more easily here. This enables RNA
polymerase to identify the start of the gene and to select the strand of
DNA to be transcribed(the antisense strand)
2) Elongation: After recognizing the promoter site, the RNA
polymerase begins to synthesize a complementary transcript of the
DNA template, starting from 5’ end of the RNA being synthesized.
Whenever adenine is present on the DNA, uracil is incorporated into

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the RNA. Whenever thymine is present on the DNA, adenine is

incorporated. Whenever G is present, C is incorporated, and vice
versa. Each ribonucleotide is incorporated one at a time into the RNA
being synthesized.
3) Termination: The RNA polymerase continues to transcribe the
antisense templet DNA strand until a termination signal is reached,
which signals the end of the message. This sequence contains a G –C-
rich region followed by an A-T-rich sequence. At this point, both
RNA polymerase and the newly synthesized RNA transcript have the
DNA template, which rewinds completely.
Types of RNA: There are three types of RNA
1) Messenger RNA (mRNA): There are single stranded polyribonucleotide
molecules which may be long or short depending on whether they code for a
large polypeptide or a small one. Generally, m-RNA are short-lived
molecules (with a half life of about 2 minutes in bacteria). They compose
~3% of cellular RNA.
Functions of mRNA: The nucleotide sequence of mRNA is complementary to the
genetic massage present in a specific region of DNA. It carries this genetic
information from DNA in the nucleus to ribosomes in the cytoplasm. Ribosomes
then use the mRNA as template to translate the genetic information into the amino
acid sequence of proteins.
2) Ribosomal RNA (rRNA): These make up about 80% of cellular RNA and
constitute upto 65% of the mass of ribosomes (the sites of protein synthesis).
r-RNA is synthesized in the nucleons present in the nucleus
Functions rRNA: r- RNA makes up the machinery or assembly –line where
proteins are synthesized.

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3) Transfer RNA (t-RNA): These compose about 15% of cellular RNA. There
are smallest of all the RNAs (73-95 nucleotides long) and are present in a
highly folded cloverleaf –like conformation, which contains double stranded
regions where its bases are paired. The t-RNA contains unusual bases. Each
of the amino acids found in proteins has at least one type of t-RNA specific
for it.
Function of tRNA: Each t-RNA recognizes and combines with specific amino
acid and carries it to the ribosomes. Each t-RNA contains a highly specific
sequence of three nucleotides called its anticodon. This anticodon is
complementary to a codon, a sequence of three ribonucleotides on m-RNA that
specifically codes for a particular amino acid.
Another class of RNA is present in all cells called small RNA. Some of
these have catalytic activity or else participate in catalytic activity alongside
proteins.
Translation (protein synthesis): This is the process in which the genetic
information encoded in an mRNA molecule is used to specify the sequence of
amino acids during synthesis of proteins.
Mechanism of translation: The stages of translation: Ribosomes are the
macromolecular assembles which constitute the machinery where proteins are
made. They consist of two units of unequal size. They translate m-RNA in the

direction, and synthesize proteins from the amino terminal end to the
carboxy-terminal end. Protein synthesis occurs in four interrelated steps.
1) Activation of amino acids: Each amino acid is activated by forming a
covalent complex with a specific t-RNA molecule. The reaction is catalysed
by enzymes called aminoacetyl – t-RNA synthetases.

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The aminoacyl t-RNA synthetases are highly specific molecules, and play a
vital role in attaching the right amino acid to the right t-RNA.

2) Initiation: the smaller subunit of a ribosome first binds the mRNA in a

precisely oriented manner. Each triplet unit (codon) on this specifies a
particular amino acid. In prokaryotes, the first codon is always AUG, which
codes for methionine. To this codon, a t-RNA molecule binds through its
anti-codon (in prokaryotes, this first t-RNA will always be attached to N-
formyl methionine). After this aminoacyl tRNA attaches, the large ribosomal
subunit binds to give the complete complex. Initiation requires energy which
is provided by the hydrolysis of ATP and GTP.
3) Elongation: At RNA carrying the second amino acid now binds to the next
codon (towards the 3’ direction of the mRNA) this requires GTP hydrolysis.
The two amino acids then react forming a peptide bond. In this process the
first amino acid (N –formyl methaonine) is transferred to the second t-RNA.
The first t-RNA is released. This reaction is catalyzed by peptidyl
transferase of the large ribosomal unit. GTP is again hydrolyzed and the
ribosome moves three bases along the 5’ 3’ direction of the m-RNA. This
process called translocation, also positions the next codon, getting it ready
for the t-RNA carrying the third amino acid. At the same time, the empty t-
RNA leaves the ribosomal complex.
The polypeptide is lengthened by forming peptide bonds between
successive amino acid units. Each amino acid is brought and put in its correct place
by its corresponding t-RNA. Each codon on m-RNA is recognized by precise
empery hydrogen bonding with the specific anti-codon of the correct t-RNA

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4) Termination and release: The process of elongation continues until a

termination codon is reached. At this point, the completed polypeptide is
released from the ribosome. GTP is again hydrolyzed and the ribosomal
subunits dissociate from the mRNA. Often, the same mRNA molecule is
translated by several ribosomes simultaneously. This increases the rate of
polypeptide synthesis. Such a group of ribosomes attached to the same
molecules of mRNA are called polysomes. The above stages in protein
synthesis are shown in below figure.

As the polypeptide is released from the ribosome, it folds up into its native,
biologically active, conformation. This is aided by proteins called molecular
chaperones. Before or after folding, some parts of the protein may be removed and

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other groups like prosthetic groups may be introduced. Post translational

modification of amino acids may also occur.
DNA fingerprinting: DNA fingerprinting (also called DNA profiling or DNA
typing) is a technique used to identify an individual from a sample of DNA by
looking at unique patterns in their DNA. It is now used for lots of purpose but
forensic is the major field in which it is used.
Principle of DNA fingerprinting: Human genome contains 3 billion bases
which are arranged in a particular sequence that gives us a unique identity. 90% of
the DNA is same in every human beings (about 90% nucleotide bases are exactly
same in human beings). DNA fingerprinting is bases upon the rest 10% difference
in the human DNA. This method is done by matching the uncommon sequence of
humans with the suspect’s unique sequence.
Applications: DNA fingerprinting has got lots of applications
1) This procedure is mostly used in forensic to indentify the criminals
2) It is also used for the paternity test
3) It is used in the study of breeding patterns of animals facing the danger of
extinction
4) It is also used in determining lineages of humans and other animals to
ascertain the process of evolution by checking the “genetic markers” which
are passed from the ancestors.
5) It is used to diagnose the genetic disorders and hereditary disorders like
hemophilia, sickle cell anemia, cystic fibrosis, etc.
6) It is also used to determine about the antibiotics to which bacteria’s are
resistant

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