By:

Sajan Maharjan
M.Pharm, 2nd year
Kathmandu University
Structure of DNA
DNA is usually a double-helix and has two strands running in opposite directions (exception: some viral
DNA are single-stranded). Each chain is a polymer of subunits called nucleotides, hence they are named
polynucleotide.

Each strand has a backbone made up of (deoxy-ribose) sugar molecules linked together by phosphate
groups. The 3' C of a sugar molecule is connected through a phosphate group to the 5' C of the next sugar.
This linkage is also called 3'-5' phosphodiester linkage. All DNA strands are read from the 5' to the 3' end
where the 5' end terminates in a phosphate group and the 3' end terminates in a sugar molecule.

Each sugar molecule is covalently linked to one of 4 possible bases (Adenine, Guanine, Cytosine
and Thymine). A and G are double-ringed larger molecules (called purines); C and T are single-ringed
smaller molecules (called pyrimidines).
In the double-stranded DNA, the two strands run in opposite directions and the bases pair up such that A
always pairs with T and G always pairs with C. The A-T base-pair has 2 hydrogen bonds and the G-C
base-pair has 3 hydrogen bonds. The G-C interaction is therefore stronger (by about 30%) than A-T, and
A-T rich regions of DNA are more prone to thermal fluctuations.

The most common DNA structure in solution is the B-DNA. Under conditions of applied force or twists
in the DNA, or under low hydration conditions, it can adopt several helical conformations, referred to as
the A-DNA, Z-DNA, S-DNA.

A-DNA

B-DNA

Z-DNA

Right-handed helix

Right-handed

Left-handed

Short and broad

Long and thin

Longer and thinner

Helix Diameter

25.5A

23.7A

18.4A

Rise / base-pair

2.3A

3.4A

3.8A

Base-pair / helical turn

~ 11

~ 10

~ 12

Helix pitch

25A

34A

Tilt of the bases

20 deg

-1 deg

47A
-9 deg

DNA Replication
DNA replication is the process by which DNA makes a copy of itself during cell division. Before a cell
can divide, it must duplicate all its DNA. Where DNA replication occurs depends upon whether the cells
is a prokaryote or a eukaryote. DNA replication occurs in the cytoplasm of prokaryotes and in the nucleus
of eukaryotes. In eukaryotes, this occurs during S phase of the cell cycle. Regardless of where DNA
replication occurs, the basic process is the same.
DNA Replication Models
There are three possible models that describe the accurate creation of the daughter chains:
1) Semiconservative Replication:
According to this model, DNA Replication would create two molecules. Each of them would be a
complex of parental and a daughter strand.
2) Conservative Replication:
According to this model, the DNA Replication process would create a brand new DNA double helix made
of two daughter strands while the parental chains would stay together.
3) Dispersive Replication:
According to this model the Replication Process would create two DNA double-chains, each of them with
parts of both parent and daughter molecules.
Among all of these models, Semiconservative DNA Replication was proved by the experiment
of Meselson - Stahl.
Steps of DNA Replication Process
DNA Replication proceeds in three enzymatically catalyzed and coordinated steps:
1. Initiation
2. Elongation
3. Termination
1. Initiation
DNA replication process is initiated at particular points in the DNA, known as "origins", which are
targeted by initiator proteins. Sequences used by initiator proteins tend to be rich in adenine and
thymine bases, because A-T base pairs have two hydrogen bonds (rather than the three formed in a CG pair) which are easier to unzip. Once the origin has been located, these initiators recruit other
proteins and form the pre-replication complex, which unzips the double-stranded DNA. Helicase is
the enzyme that splits the two strands and the structure that is created is known as "Replication Fork".

2. Elongation
The next most important step of DNA Replication is the binding of RNA Primase in the initiation
point of the 3'-5' parent chain. RNA Primase can attract RNA nucleotides which bind to the DNA

nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the
primers (starters) for the binding of DNA nucleotides.

The elongation process is different for the 5'-3' and 3'-5' template.
a) 5'-3' Template: The 3'-5' proceeding daughter strand, that uses a 5'-3' template- is called leading
strand because DNA Polymerase can read the template and continuously add nucleotides.
b) 3'-5'Template: The 3'-5' template cannot be read by DNA Polymerase. The replication of this
template is complicated and the new strand is called lagging strand. In the lagging strand the RNA
Primase adds more RNA Primers. DNA polymerase reads the template and lengthens the bursts. The
gap between two RNA primers is called "Okazaki Fragments"

In the lagging strand the DNA Pol I -exonuclease reads the fragments and removes the RNA Primers.
The gaps are closed with the action of DNA Polymerase that add complementary nucleotides to the
gaps and DNA Ligase adds phosphate in the remaining gaps of the phosphate - sugar backbone.
Each new double helix is consisted of one old and one new chain. So, this model of replication is
called semiconservative replication.
3. Termination
The last step of DNA Replication is the Termination. This process starts when the DNA Polymerase
reaches to an end of the strands. When the RNA primer is removed from lagging strand, it is not
possible for the DNA Polymerase to seal the gap. So, the end of the parental strand where the last
primer binds, is not replicated. These ends of DNA consists of noncoding DNA that contains repeat
sequences and are called telomeres. As a result, a part of the telomere is removed in every cycle of
DNA Replication.
The DNA Replication is not completed before a mechanism of repair fixes possible errors caused
during the replication. Enzymes like nucleases remove the wrong nucleotides and the DNA
Polymerase fills the gaps.

Transcription

Transcription is the biochemical process of transferring the information in a DNA sequence to an RNA
molecule. The RNA molecule can be the final product, or in the case of messenger RNA (mRNA), it can
be used in the process of translation to produce proteins. RNA Polymerase is a protein complex that
performs the main job of reading a DNA template and synthesizing RNA, but accessory proteins are also
needed. Transcription has three major phases: Initiation, elongation and termination.
Initiation
Just before initiation, RNA polymerase and accessory proteins bind to a DNA molecule upstream of the
initiation point. The DNA is unwound to separate and expose the strand to be transcribed. Then, the RNA
polymerase complex binds to a promoter sequence, which establishes initiation of transcription.
Polymerase begins to synthesize a strand of RNA complementary to one side of the DNA strand, moving
into the coding sequence portion of the gene being transcribed.

Unwinding of DNA

Initiation of Transcription

Elongation
During elongation, a lengthening RNA molecule is produced by DNA polymerase as it reads the DNA
triplet code on the template strand. The polymerase will continue reading the template until it reaches a
sequence that provides a signal indicating that transcribed region is at an end. Another RNA polymerase
can attach to the promoter to begin synthesizing another RNA before the first one is finished.

Termination
Termination of transcription is triggered when the RNA polymerase encounters a particular DNA
sequence, causing the polymerase to lose affinity for the DNA template. At this point, RNA polymerase
disengages from the DNA and the RNA molecule is released for translation or post-transcriptional
processing.

Transcription Factors
Other proteins besides RNA polymerase are required for transcription. These proteins are called
transcription factors. They may bind to RNA polymerase, interact with other transcription factors, or bind

to DNA directly to affect transcription. Transcription factors are required for proper assembly of the
initiation complex, and have important functions in elongation and termination.
Regulation of Transcription
The efficiency and degree to which transcription occurs is regulated by the aforementioned transcription
factors as well as DNA binding proteins. Suppressor proteins attach to DNA to block initiation,
preventing certain genes from being transcribed. Other molecules can interact with suppressors, causing
them to leave their DNA binding sites, allowing transcription to proceed.
Translation
Translation is the process of turning the coded message in the messenger RNA into the final protein chain.
Translation is the final step on the way from DNA to protein. It is the synthesis of proteins directed by a
mRNA template. The information contained in the nucleotide sequence of the mRNA is read as three
letter words (triplets), called codons. Each word stands for one amino acid.
During translation, amino acids are linked together to form a polypeptide chain which will later be folded
into a protein. The translation is dependent on many components. Some of them are:
1. Ribosome: It is the cellular factory responsible for the protein synthesis. It consists of two
different subunits, one small and one large and is built up from rRNA and proteins. Inside the
ribosome the amino acids are linked together into a chain through multiple biochemical reactions.
2. tRNA: It is a specialised RNA molecule that carries an amino acid at one end and has a triplet of
nucleotides, an anticodon, at the other end. The anticodon of a tRNA molecule can basepair, i.e
form chemical bonds, with the mRNA's three letter codon.Thus the tRNA acts as the translator
between mRNA and protein by bringing the specific amino acid coded for by the mRNA codon.
3. mRNA: mRNA is the product of transcription.It is a single-strand of ribonucleotides that is
complementary to its gene template. The purpose of mRNA is to carry the genetic code from
DNA to the ribosome for translation. mRNA is read in a series of triplets called codons. For
example, the mRNA sequence AUGAAGCACUAC has four codons. Each codon corresponds to
one amino acid.
In the above code: AUG codes for the amino acid MET, AAG codes for Lys, CAC codes for His
and UAC codes for Tyr.
The dictionary of the genetic code tells us of the 20 amino acids :

Steps in translation process

The translation process is divided into three steps:
1. Initiation:

When a small subunit of a ribosome charged with a tRNA+the amino acid methionine encounters
an mRNA, it attaches and starts to scan for a start signal. When it finds the start sequence AUG,
the codon (triplet) for the amino acid methionine, the large subunit joins the small one to form a
complete ribosome and the protein synthesis is initiated.
Initiation Step 1.
The mRNA joins to the small ribosomal unit at the 5' untranslated region. This binds to a special
binding site on the small ribosomal subunit.
The large ribosomal subunit has 3 binding sites, E, P, and A.

Initiation Step 2.
The large ribosomal subunit attaches to the small subunit such that the first codon is aligned at the
P binding site.

Initiation step 3.
A tRNA carrrying the amino acid methionine attaches to the start codon (AUG) on the
messaenger RNA. This inititates elongation.

2. Elongation:
A new tRNA+amino acid enters the ribosome, at the next codon downstream of the AUG codon.
If its anticodon matches the mRNA codon it basepairs and the ribosome can link the two
aminoacids together. If a tRNA with the wrong anticodon enters the ribosome, it can not basepair

with the mRNA and is rejected. The ribosome then moves one triplet forward and a new
tRNA+amino acid can enter the ribosome and the procedure is repeated.
Elongation step 1.
Attachment of first amino acid carrying tRNA to A binding site.
A tRNA and its amino acid attaches to the A binding site.

Elongation Step 2.
Peptide bond formation between the met and the amino acid carried at the A binding site.
Polypeptide chain now becomes: Met Thr

Elongation Step 3.
Ribosome moves in the 3' direction down the messenger RNA by three bases or one codon
shifting the tRNA and polypeptide chain to the P Binding site. The A binding site opens and a
vacant tRNA is in the E binding site.

Elongation Step 4.
tRNA is ejected from the E binding site.

Elongation Step 1 - 4 is repeated until stop codon is encountered.

3. Termination:
When the ribosome reaches one of three stop codons, for example UGA, there are no
corresponding tRNAs to that sequence. Instead termination proteins bind to the ribosome and
stimulate the release of the polypeptide chain (the protein), and the ribosome dissociates from the
mRNA. When the ribosome is released from the mRNA, its large and small subunit dissociate.
The small subunit can now be loaded with a new tRNA+methionine and start translation once
again. Some cells need large quantities of a particular protein. To meet this requirement they
make many mRNA copies of the corresponding gene and have many ribosomes working on each
mRNA. After translation the protein will usually undergo some further modifications before it
becomes fully active.
Termination Step 1.
The polypeptide chain is at the P site. The stop codon at the A site.

Termination Step 2.
A Release factor protein binds to the stop codon at the A binding site.

Termination Step 3.
Release factor protein initiates separation of polypeptide chain: Met-Thr-His-Asp-Gly

Termination Step 4.
Separation of translation machinary. Polypeptide chain may go to cytoplasm for further
processing.