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Molecular Biology

Molecular biology
Field of science concerned with the chemical
structures and processes of biological phenomena
at the molecular level. Having developed out of the
related fields of biochemistry, genetics, and
biophysics, the discipline is particularly concerned
with the study of proteins, nucleic acids, and
enzymes. In the early 1950s, growing knowledge of
the structure of proteins enabled the structure of
DNA to be described. The discovery in the 1970s of
certain types of enzymes that can cut and
recombine segments of DNA (recombination) in the
chromosomes of certain bacteria made
recombinant-DNA technology possible. Molecular
biologists use that technology to isolate and modify
specific gene
Molecular Biology
• Nucleic acids and nucleoprotein structure.
• Replication.
• Transcription.
• Regulation of gene expression.
• Restriction enzymes & its function in DNA technology.
• Gene cloning .
• Production of recombinant plasmid.
• Construction of genomic and DNA libraries.
• Analyzing & sequencing cloned DNA.
• Analysis of specific nucleic acids in complex mixtures
• polymerase chain reaction (PCR),mutation .
Deoxyribonucleic acid
(DNA) is a nucleic acid that contains the genetic
instructions used in the development and
functioning of all known living organisms. The main
role of DNA molecules is the long-term storage of
information and DNA is often compared to a set of
blueprints, since it contains the instructions needed
to construct other components of cells, such as
proteins and RNA molecules. The DNA segments
that carry this genetic information are called genes,
but other DNA sequences have structural purposes,
or are involved in regulating the use of this genetic
information.
• In living organisms, DNA does not usually
exist as a single molecule, but instead as a
tightly-associated pair of molecules. These
two long strands entwine like vines, in the
shape of a double helix. The nucleotide
repeats contain both the segment of the
backbone of the molecule, which holds the
chain together, and a base, which interacts
with the other DNA strand in the helix. In
general, a base linked to a sugar is called a
nucleoside and a base linked to a sugar and
one or more phosphate groups is called a
nucleotide. If multiple nucleotides are
linked together, as in DNA, this polymer is
referred to as a polynucleotide.
• Nucleotide structure
• A nucleotide is composed of a nucleobase (nitrogenous base), a
five-carbon sugar (either ribose or 2'-deoxyribose), and one to
three phosphate groups. Together, the nucleobase and sugar
comprise a nucleoside. The phosphate groups form bonds with
either the 2, 3, or 5-carbon of the sugar, with the 5-carbon site
most common. Cyclic nucleotides form when the phosphate group
is bound to two of the sugar's hydroxyl groups. Ribonucleotides are
nucleotides where the sugar is ribose, and deoxyribonucleotides
contain the sugar deoxyribose. Nucleotides can contain either a
purine or pyrimidine base.
• Nucleic acids are polymeric macromolecules made from nucleotide
monomers. In DNA, the purine bases are adenine and guanine,
while the pyrimidines are thymine and cytosine. RNA uses uracil in
place of thymine.
• Synthesis
• Nucleotides can be synthesized by a variety of means both in vitro
and in vivo. In vivo, nucleotides can be synthesised de novo or
recycled through salvage pathways. Nucleotides undergo
breakdown such that useful parts can be reused in synthesis
reactions to create new nucleotides. In vitro, protecting groups
may be used during laboratory production of nucleotides. A
purified nucleoside is protected to create a phosphoramidite,
which can then be used to obtain analogues not found in nature
and/or to synthesize an oligonucleotide
DNA's duplex nature
• DNA is normally double-stranded. The sequences of
the two strands are related so that an A on one
strand is matched by a T on the other strand;
likewise, a G on one strand is matched by a C on the
other strand. Thus, the fraction of bases in an
organism's DNA that are A is equal to the fraction of
bases that are T, and the fraction of bases that are G
is equal to the fraction of bases that are C. For
example, if one-third of the bases are A, one-third
must be T, and because the amount of G equals the
amount of C, one-sixth of the bases will be G and
one-sixth will be C. The importance of this
relationship, termed Chargraff's rules, was
recognized by Watson and Crick, who proposed that
the two strands form a double helix with the two
strands arranged in an antiparallel fashion,
interwound head-to-tail
• In a double helix the direction of the nucleotides
in one strand is opposite to their direction in the
other strand. This arrangement of DNA strands is
called antiparallel. The asymmetric ends of DNA
strands are referred to as the 5′ (five prime) and
3′ (three prime) ends.
• One of the major differences between DNA and
RNA is the sugar, with 2-deoxyribose being
replaced by the alternative pentose sugar ribose
in RNA.
• Usually,we read nucleic acid sequences of DNA in
a 5′ to 3′ direction, so a DNA dinucleotide of (51)
adenosine-guanosine (31) is read as AG.
• The complementary sequence is CT, because
both sequences are read in the 5′ to 3′ direction.
The terms 5′ and 3′ refer to the numbers of the
carbons on the sugar portion of the nucleotide
(the base is attached to the 1′ carbon of the
sugar).
•Chemically, DNA is a long polymer of simple units called nucleotides, with a
backbone made of sugars and phosphate groups joined by ester bonds.
Attached to each sugar is one of four types of molecules called bases. It is
the sequence of these four bases along the backbone that encodes
information.
Nucleotides

Adenosine monophosphate Adenosine diphosphate Adenosine triphosphate


AMP ADP ATP

Guanosine monophosphate Guanosine diphosphate Guanosine triphosphate


GMP GDP GTP

Thymidine monophosphate Thymidine diphosphate Thymidine triphosphate


TMP TDP TTP
Uridine monophosphate Uridine diphosphate Uridine triphosphate
UMP UDP UTP

Cytidine monophosphate Cytidine diphosphate Cytidine triphosphate


CMP CDP CTP
Deoxynucleotides

Deoxyadenosine monophosphate Deoxyadenosine diphosphate Deoxyadenosine triphosphate


dAMP dADP dATP

Deoxyguanosine monophosphate Deoxyguanosine diphosphate Deoxyguanosine triphosphate


dGMP dGDP dGTP

thymidine monophosphate thymidine diphosphate thymidine triphosphate


TMP TDP TTP

Deoxyuridine monophosphate Deoxyuridine diphosphate Deoxyuridine triphosphate


dUMP dUDP dUTP

Deoxycytidine monophosphate Deoxycytidine diphosphate Deoxycytidine triphosphate


dCMP dCDP dCTP
Pyrimidine ribonucleotides
Pyrimidine nucleotide synthesis starts with the formation
of carbamoyl phosphate from glutamine and CO2. The
cyclisation reaction between carbamoyl phosphate reacts
with aspartate yielding orotate in subsequent steps.
Orotate reacts with 5-phosphoribosyl α-diphosphate
(PRPP) yielding orotidine monophosphate (OMP) which is
decarboxylated to form uridine monophosphate (UMP). It
is from UMP that other pyrimidine nucleotides are
derived. UMP is phosphorylated to uridine triphosphate
(UTP) via two sequential reactions with ATP. Cytidine
monophosphate (CMP) is derived from conversion of UTP
to cytidine triphosphate (CTP) with subsequent loss of
two phosphates
Nucleotides function in cell metabolism
Purine ribonucleotides
The atoms which are used to build the purine
nucleotides come from a variety of sources:
The de novo synthesis of purine nucleotides by
which these precursors are incorporated into the
purine ring, proceeds by a 10 step pathway to the
branch point intermediate IMP, the nucleotide of
the base hypoxanthine. AMP and GMP are
subsequently synthesized from this intermediate
via separate, two step each, pathways. Thus
purine moieties are initially formed as part of the
ribonucleotides rather than as free bases.
Synthesis Purine ribonucleotides
By using a variety of isotopically labeled
compounds it was demonstrated that the
sources of the atoms in purines are as follows:

The biosynthetic origins of purine ring atoms

N1 arises from the amine group of Asp


C2 and C8 originate from formate
N3 and N9 are contributed by the amide group of Gln
C4, C5 and N7 are derived from Gly
-
C6 comes from HCO3 (CO2)
Deoxyribonucleic acid (DNA)

DNA is a long polymer made from repeating units called nucleotides.[The DNA chain is 22 to
26 Angstroms' wide (2.2 to 2.6 nanometres), and one nucleotide unit is 3.3 Ångstroms
(0.33 nanometres) long. Although each individual repeating unit is very small, DNA polymers can be
enormous molecules containing millions of nucleotides. For instance, the largest human
chromosome, chromosome number 1, is 220 million base pairs long.
Major and minor grooves
The double helix is a right-handed spiral. As the
DNA strands wind around each other, they leave
gaps between each set of phosphate backbones,
revealing the sides of the bases inside
Two of these grooves twisting around the surface
of the double helix: one groove, the major groove,
is 22 Å wide and the other, the minor groove, is
12 Å wide. The narrowness of the minor groove
means that the edges of the bases are more
accessible in the major groove. As a result,
proteins like transcription factors that can bind to
specific sequences in double-stranded DNA usually
make contacts to the sides of the bases exposed in
the major groove
Base pairing
Each type of base on one strand forms a bond with
just one type of base on the other strand. This is
called complementary base pairing. Here, purines
form hydrogen bonds to pyrimidines, with A
bonding only to T, and C bonding only to G. This
arrangement of two nucleotides binding together
across the double helix is called a base pair. In a
double helix, the two strands are also held together
via forces generated by the hydrophobic effect and
pi stacking, which are not influenced by the
sequence of the DNA. As hydrogen bonds are not
covalent, they can be broken and rejoined relatively
easily. The two strands of DNA in a double helix can
therefore be pulled apart like a zipper, either by a
mechanical force or high temperature. As a result of
this complementarity, all the information in the
double-stranded sequence of a DNA helix is
duplicated on each strand, which is vital in DNA
replication. Indeed, this reversible and specific
interaction between complementary base pairs is
critical for all the functions of DNA in living
organisms.
The two types of base pairs
form different numbers of
hydrogen bonds, AT forming
two hydrogen bonds, and GC
forming three hydrogen
bonds. The GC base pair is
therefore stronger than the AT
base pair. As a result, it is both
the percentage of GC base
pairs and the overall length of
a DNA double helix that
determine the strength of the
association between the two
strands of DNA.
Long DNA helices with a high GC content have stronger-
interacting strands, while short helices with high AT content
have weaker-interacting strands.
 Parts of the DNA double helix that need to separate easily, such
as the TATAAT Pribnow box in bacterial promoters, tend to
have sequences with a high AT content, making the strands
easier to pull apart.
Sense and antisense
A DNA sequence is called "sense" if its sequence is the same as
that of a messenger RNA copy that is translated into protein.
The sequence on the opposite strand is complementary to the
sense sequence and is therefore called the "antisense" sequence.
Since RNA polymerases work by making a complementary copy
of their templates, it is this antisense strand that is the template
for producing the sense messenger RNA. Both sense and
antisense sequences can exist on different parts of the same
strand of DNA (i.e. both strands contain both sense and
antisense sequences).
Biological molecules that prefer to form strands. Wilkins
worked on the DNA project with Rosalind Franklin, who
took the X-ray photograph that gave Watson and Crick their
eureka moment. He then spent almost 10 years rigorously
verifying that breakthrough.

Linking number : in topology, the total number of


times one strand of the DNA double helix winds
around the other in a right hand direction, given a
DNA molecule with constrained ends. 2 molecules
differing only in linking number are topoisomers.
Writhing number (W) : in topology, the number of
superhelical turns in a DNA molecule with
constrained ends
Alternative double-helical structures
DNA exists in several possible conformations.
The conformations so far identified are: A-DNA,
B-DNA, C-DNA, D-DNA, E-DNA,H-DNA, L-DNA, P-
DNA, and Z-DNA
However, only A-DNA, B-DNA, and Z-DNA have
been observed in naturally occurring biological
systems
Which conformation DNA adopts depends on the
sequence of the DNA, the amount and direction
of supercoiling, chemical modifications of the
bases and also solution conditions, such as the
concentration of metal ions and polyamines
The A -DNA is a wider right-handed
spiral, with a shallow and wide minor
groove and a narrower and deeper major
groove. The A form occurs under non-
physiological conditions in dehydrated
samples of DNA, while in the cell it may be
produced in hybrid pairings of DNA and
RNA strands, as well as in enzyme-DNA
complexes

B-DNA : the usual double helical structure


assumed by double-stranded DNA; see
illustration at deoxyribonucleic acid.

Z-DNA : a form of DNA in which the


phosphate groups form a dinucleotide
repeating unit zigzagging up a left-handed helix
with a single, deep groove; it is particularly
likely to occur in stretches of alternating
From left to right, the structures of A, B
purines and pyrimidines and Z DNA
spacer DNA : the nucleotide sequences occurring
between genes, in eukaryotes often long and
including many repetitive sequences; particularly,
the DNA occurring between the genes encoding
ribosomal RNA.

complementary or copy DNA (cDNA) : synthetic


DNA transcribed from a specific RNA through the
reaction of the enzyme (reverse transcriptase).

nuclear DNA (nDNA) : the DNA of the


chromosomes found in the nucleus of a eukaryotic
cell.
Repetitive DNA : nucleotide sequences occurring
multiply within a genome; they are characteristic
of eukaryotes and generally do not encode
polypeptides. Sequences may be clustered or
dispersed, and repeated moderately (10 to 104
copies per genome) to highly (>106 copies per
genome). Moderately repetitive DNA sequences
encode some structural genes for ribosomal RNA
and histones; highly repetitive sequences are
mostly satellite DNA
Satellite DNA : short, highly repeated DNA
sequences found in eukaryotes, usually in
clusters in constitutive heterochromatin and
generally not transcribed
Mitochondrial DNA (mtDNA) : the DNA of the
mitochondrial chromosome, existing in several thousand
copies per cell and inherited exclusively from the
mother. Its code differs both from that of nuclear DNA
and from that of any present day prokaryote, and it
evolves 5 to 10 times more rapidly than nuclear DNA.

Recombinant DNA : a DNA molecule composed of linked


sequences not normally occurring within the same
molecule, such as a bacterial plasmid into which has
been inserted a segment of viral DNA.

Single copy DNA (scDNA) : nucleotide sequences present


once in the haploid genome, as are the majority of the
gene sequences encoding polypeptides in eukaryotes
THE CENTRAL DOGMA
OF MOLECULAR BIOLOGY

( THE BIOINFORMATION
THEORY)
Central Dogma of Biology
Replication
• Replication
• Chromosomes are located in the nucleus of a cell.  DNA
must be duplicated in a process called replication before a
cell divides.  The replication of DNA allows each daughter
cell to contain a full complement of chromosomes.   

• DNA Replication:
• Semiconservative Model of DNA Replication
After Watson and Crick proposed the double helix model of
DNA, three models for DNA replication were proposed:
conservative, semiconservative, and dispersive. The
semiconservative model was proved to be the correct one
Semiconservative DNA replication
The two strands in the double helix
separate, and then each strand serves as
template for the synthesis of a new
(complementary) strand. After
replication has been completed, each of
the two duplexes has one old and one
newly synthesized strand.
and dispersive modes of replication do
not make much sense, and are not
supported by experiments.
Eukaryotic DNA replication is
semiconservative
 Eukaryotic DNA replicates Semiconservatively
 by the Taylor, Woods, and Hughes experiment
in 1958.
 They labeled DNA with 3H-T, treated the roots
of Fava bean with Colchicin, fixed and prepared
for microscopy. At the first metaphase, after
labeling at interphase, both chromatids of each
chromosome ere labeled, whereas at the
second metaphase only one chromatide was
labeled
How does this show semiconservative
replication?
The DNA double helix and genetic replication

• Because an A on one strand must base-pair with a T on the


other strand, if the two strands are separated, each single
strand can specify the composition of its partner by acting as a
template.
• The DNA template strand does not carry out any enzymatic
reaction but simply allows the replication machinery (an
enzyme) to synthesize the complementary strand correctly.
• This dual-template mechanism is termed semi-conservative,
because each DNA after replication is composed of one
parental and one newly synthesized strand. Because the two
strands of the DNA double helix are interwound, they also
must be separated by the replication machinery to allow
synthesis of the new strand. Figure 3 shows this replication.
Features of DNA replication
• Bidirectional. Starts at specific sites (origins) and moves
• in opposite directions using two replication “forks”.
• • Semi-discontinuous. One strand (leading) replicates
continuously and the other (lagging) discontinuously
• • In the 5’ - 3’ direction. Enzymes (DNA polymerases) can only add
a nucleotide to a free OH group at the 3'-end of a growing chain
The double-stranded DNA shown above is unwinding and ready for
replication. Note the antiparallel nature of the strands; that is, the 5'
to 3' orientation of the top strand and the 3' to 5' orientation of the
complementary bottom strand.

A. The DNA is already partially unwound to


form a replication fork.

B. On the bottom template strand, primase


synthesizes a short RNA primer in the 5' to 3'
direction.

C. Primase leaves, and DNA polymerase adds


DNA nucleotides to the RNA primer in the 5'
to 3' direction. In E. coli the enzyme used is
DNA polymerase III. This new DNA is called
the leading strand because it is being made in
the same direction as the movement of the
replication fork.
Enzymes and Proteins in DNA Replication

• A large number of enzymes and other proteins


are involved in the synthesis of new DNA at a
replication fork.
• Alternative DNA polymerase:
• This DNA polymerase replaces the RNA primer with DNA. This
is a different type of DNA polymerase from the main DNA
polymerase which synthesizes DNA on a DNA template.
• In E. coli the main enzyme is DNA polymerase III and the
enzyme that replaces the RNA primer with DNA is DNA
polymerase I.
• When the RNA primer has been replaced with DNA, there is a
gap between the two Okazaki fragments and this is sealed by
DNA ligase
DNA ligase:
• DNA ligase seals the gap left between Okazaki fragments after the
primer is removed. As the Okazaki fragments are joined, the new
lagging strand becomes longer and longer.

• DNA polymerase:
• Location: On the template strands.
• Function: Synthesizes new DNA in the 5' to 3' direction using the base
information on the template strand to specify the nucleotide to insert
on the new chain. Also does some proofreading; that is, it checks that
the new nucleotide being added to the chain carries the correct base
as specified by the template DNA. If an incorrect base pair is formed,
DNA polymerase can delete the new nucleotide and try again.
• • Lagging Strand:
• The new DNA strand made discontinuously in the
direction opposite to the direction in which the
replication fork is moving.

• • Leading strand:
• The new DNA strand made continuously in the
same direction as movement of the replication
fork.

• • Okazaki fragment:
• Location: On the template strand which dictates
new DNA synthesis away from the direction of
replication fork movement.
• Function: A building block for DNA synthesis of
the lagging strand. On one template strand, DNA
polymerase synthesizes new DNA in a direction
away from the replication fork movement.
Because of this, the new DNA synthesized on that
template is made in a discontinuous fashion; each
segment is called an Okazaki fragment.
• • Helicase:
• Location: At the replication fork.
• Function: Unwinds the DNA double
helix.
• • Primase:
• Location: Wherever the synthesis of a
new DNA fragment is to commence.
Function: DNA polymerase cannot start
the synthesis of a new DNA chain, it can
only extend a nucleotide chain primer.
• Single-strand binding (SSB) proteins:
Primase synthesizes a short RNA chain
Location: On single-stranded DNA
that is used as the primer for DNA
near the replication fork.
synthesis by DNA polymerase.
Function: Binds to single-stranded
DNA to make it stable.
• Overall direction of
replication (movement
of replication fork):
The direction of replication i.e.,
the direction in which the
replication fork moves as the
DNA double helix unwinds.
• Parent DNA:
The parental DNA double helix
that will be unwound and used
as the template for new DNA
synthesis.

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